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The fMRI signature of acute catatonic state and its response to benzodiazepines \| medRxiv medRxiv - The Preprint Server for Health Sciences The fMRI signature of acute catatonic state and its response to benzodiazepines View ORCID Profile Pravesh Parekh , Anirban Gozi , Venkata Senthil Kumar Reddi , Jitender Saini , View ORCID Profile John P. John doi: https://doi.org/10.1101/2021.03.23.21253765 Abstract We report for the first time, functional MRI markers of the acute retarded catatonic state and its response to benzodiazepines. In this cross-sectional MRI study, we have compared the resting state whole-brain, within-network and seed (left precentral gyrus)-to-voxel connectivity, as well as cortical complexity between a sample of patients in acute retarded catatonic state ( n= 15) and a demographically-matched healthy control sample ( n= 15). Additionally, we examined whether the above variables were different between responders ( n= 9) and non-responders ( n= 6) to lorazepam. Acute retarded catatonia was characterized by reduced functional connectivity, most robustly within the sensorimotor network, diffuse long-range hyperconnectivity, and seed (left precentral gyrus)-to-voxel hyperconnectivity in the frontoparietal and cerebellar regions. The lorazepam responders showed long-range as well as seed-to-voxel functional hyperconnectivity in comparison to the non-responders. Seed (left precentral gyrus)-to-voxel connectivity was positively correlated to the catatonia motor ratings. The catatonia sample showed a cluster of reduced vertex-wise cortical complexity in the right insular cortex and contiguous areas. We have identified neuroimaging markers that characterize the acute retarded catatonic state and predict treatment response. We discuss how these novel findings have important translational implications for understanding the pathophysiology of catatonia and for predicting treatment response to benzodiazepines. Introduction Catatonia is a complex syndrome of severe psychomotor dysregulation seen in various neuropsychiatric conditions. 1 , 2Irrespective of the nature of the underlying primary neuropsychiatric disorder, the acute retarded catatonic syndrome shows a remarkable similarity in clinical presentation across all patients suffering from the condition. Moreover, it is a unique psychiatric syndrome that dramatically responds fully to acute treatment in a matter of hours to days, and therefore offers a very promising disease model for studying neuroimaging markers that predict treatment response. However, functional MRI (fMRI) studies in catatonia have so far been reported only in patients following recovery, or in stable patients having mild catatonic symptoms 2 , 3(see Supplementary Table 1). This is likely due to the immense challenges involved in acquiring fMRI scans in the short time window between clinical presentation of the patient to the emergency psychiatry services and recovery from catatonia following initiation of treatment. We aimed to study structural and resting state fMRI abnormalities in patients with acute retarded catatonia presenting to the psychiatric emergency services. We compared resting state functional connectivity (rs-FC) between the acute retarded catatonia sample and a demographically matched healthy sample; and attempted to link these with treatment response to benzodiazepines. There have been no published resting fMRI studies in acutecatatonia so far 2(refer Supplementary Table 1 for a summary of previous functional brain imaging studies in catatonia). Nonetheless, since acute retarded catatonia is a condition typically characterized by severe psychomotor retardation but somewhat paradoxically responds dramatically to the GABA-ergic agonists, benzodiazepines, we hypothesized that the acute catatonia sample will have higher rs-FC in comparison to the healthy control sample. We also explored the differences in rs-FC between responders to benzodiazepines and non-responders, and the relationship between seed-to-voxel rs-FC from the left precentral gyrus (PCG) and severity of motor symptoms. Structural imaging studies in catatonia have reported inconsistent findings (for review, see). 2A measure of cortical folding complexity known as cortical surface complexity 4capable of detecting relatively small local differences, has been proposed as a temporally stable neurodevelopmental marker of cortical shape and abnormal gyrification in various neuropsychiatric disorders. 4 – 9. We therefore examined alterations in regional brain volumes (using voxel-based morphometry, VBM) and surface complexity in our sample of patients with acute retarded catatonia in comparison to the healthy control sample. Materials and methods Study samples In this first fMRI study in acute retarded catatonia, we recruited a sample of 15 right-handed patients (8 female patients) from the psychiatric emergency services of the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India, after obtaining written informed consent on behalf of the patients from the accompanying caregiver/legally authorized representative (following recovery from catatonia, written informed consent was obtained from patients as well) with the full approval of the NIMHANS Ethics Committee. Within this sample of patients, nine patients responded to lorazepam (‘LZM’ subgroup) while the remaining six did not; and required electroconvulsive therapy (ECT) for resolution of their catatonia state (‘ECT’ subgroup). We additionally recruited an age-matched sample of 15 right-handed healthy subjects (8 female subjects; ‘HS’ group). A detailed description of patient recruitment, establishment of diagnoses as per Diagnostic and Statistical Manual (DSM)-5 10criteria, socio-demographic details (Supplementary Table 2), and subject-wise clinical details (Supplementary Table 3) is provided in the Supplementary material. MRI Data acquisition Structural brain imaging (CT/MRI) is an integral part of the clinical evaluation of patients who present to the psychiatric emergency services with catatonia. 11We aimed at acquiring the MRI scan while the patients were in the acute retarded catatonic state, and wherever possible, prior to initiation of treatment for catatonia. For seven out of 15 patients, we were able to schedule the MRI acquisition promptly before the patients were initiated on lorazepam; for the remaining eight patients, we were able to perform the MRI acquisition only after patients were initiated on treatment with lorazepam, typically due to scanner unavailability at a short notice. The initiation of treatment was never delayed on account of the patient’s participation in the study. The baseline Bush Francis Catatonia Rating Scale 12(BFCRS) rating was performed within an hour prior to the MRI acquisition for all the patients (mean score: 21.07 ± 5.69; range: 12-31; Supplementary Tables 2 and 3), indicating that all patients were in acute retarded catatonic state at the time of MRI acquisition. The MRI of the patients and healthy subjects (except the last four healthy subjects—see below) were acquired on a 3T Philips Achieva scanner and consisted of a high-resolution T1-weighted scan and an eyes-open resting-state echo planar imaging (EPI) blood oxygenation level-dependent (BOLD) fMRI scan. The data of the last four (out of 15) healthy participants were acquired on a 3T Philips Ingenia CX scanner with a slightly different set of acquisition parameters. This was necessitated due to a scheduled hardware upgradation of the Achieva scanner, before data acquisition for the present study could be completed (see Supplementary Table 4 for key acquisition parameters). Preprocessing, quality check, and analyses We converted the Digital Imaging and Communications in Medicine (DICOM) images to The Neuroimaging Informatics Technology Initiative (NIfTI) format using dcm2niix 13v20181125 (https://github.com/rordenlab/dcm2niix). We reviewed the T1-weighted structural images to ensure that there were no gross anatomical abnormalities and MR artefacts. For functional images, we performed motion correction and then identified the time points with excessive motion; these time points were censored during the denoising step by modelling them as regressors. The two groups did not differ significantly in the total number of detected outliers. All analyses were performed on MATLAB R2016a (MathWorks, Natick, USA;https://www.mathworks.com). Structural images were processed using the Computational Anatomy Toolbox 14(http://dbm.neuro.uni-jena.de/cat; version 1727) with Statistical Parametric Mapping (SPM;https://www.fil.ion.ucl.ac.uk/spm; version 7771); functional images were processed using the default pipeline implemented in Conn functional connectivity toolbox 15version 18b with SPM version 7487 in the background (see Supplementary materials for preprocessing details, quality check, and analyses). Whole brain functional connectivity Within network functional connectivity We examined between-group differences in functional connectivity within the sensorimotor, salience, frontoparietal, cerebellar, and subcortical networks which encompass the critical brain regions that have been linked to hypokinetic catatonia. 3The sensorimotor, salience, and frontoparietal networks were defined using the networksatlas in Conn, while the cerebellar and subcortical networks were defined using the atlasparcellation scheme in Conn (see Supplementary Table 6 for ROIs in each network). For each of these networks, we computed the connectivity matrices and tested the same set of hypotheses as in the previous section. Since we performed a test for five networks separately, we applied an additional Bonferroni correction for multiple comparisons by testing the p-FDR values against a threshold of 0.01. Seed (left precentral gyrus)-to-voxel connectivity and regression analyses Motor disturbances are considered by most authors as the central feature of catatonia. 3In order to examine the specific abnormalities of rs-FC of the primary motor cortex with other brain regions, we compared seed-to-voxel connectivity between the study groups with the left PCG as the seed (see Supplementary Fig. 1 for ROI definition). We tested the same set of hypotheses as in the whole-brain functional connectivity section using the same statistical threshold. We also performed a linear regression analysis to examine the relationship between the BFCRS motor sub-score on day one (baseline) and the left PCG-based seed-to-voxel connectivity. For this analysis, the day one motor score, which reflects the severity of the motor dysfunction at the time of fMRI acquisition, was the predictor of the seed-to-voxel connectivity; the statistical threshold was the same as before. Fig. 1. Altered resting state functional connectivity and cortical complexity in acute retarded catatonia (CAT) ( n = 15) compared to healthy subjects (HS) ( n = 15); and between the ‘lorazepam responders (LZM)’ ( n = 9) and ‘lorazepam non-responders (ECT)’ ( n = 6). (A) In this panel, we present the whole-brain FC differences between the CAT and HS samples at seed-level p-FDR < 0.05; we found widespread increased FC in long-range connections originating in the sensorimotor, salience, frontoparietal, temporal, and cerebellar regions; and decreased FC in the regional and/or interhemispheric connections of the sensorimotor, temporo-parietal and cerebellar regions. (B) In this panel, we present the whole-brain FC differences between the LZM and ECT samples at seed-level p-FDR < 0.05; we found increased FC in the LZM sub-group compared to the ECT sub-group. (C) In this panel, we present the differences in seed-to-voxel FC from the left precentral gyrus between the CAT and HS samples at voxel-wise p-uncorrected < 0.001 and cluster-wise p-FDR < 0.05 24; we found four clusters of increased connectivity in the catatonia group; a single cluster of decreased connectivity noted in the figure was localized to the unlabelled region of the atlas. (D) In this panel, we present the differences in seed-to-voxel FC from left precentral gyrus between the LZM and ECT sub-samples at voxel-wise p-uncorrected < 0.001 and cluster-wise p-FDR < 0.05 24; we found increased FC in the LZM sub-group as compared to ECT sub-group. (E) In this panel, we present the within-network FC differences of the sensorimotor, salience, frontoparietal, and cerebellar networks between the CAT and HS samples at seed-level p-FDR < 0.05 for each network; we found decreased FC within these networks in the catatonia group that comprised of brain regions which have previously been implicated in catatonia 3; we did not observe any significant differences within the subcortical network between the two groups, nor were there any significant differences between the LZM and ECT sub-groups. (F) In this panel, we present the vertex-wise comparison of cortical complexity between HS and CAT samples (HS > CAT contrast) at p< 0.05, FWE corrected (using a non-parametric threshold-free cluster enhancement approach); we found lower cortical complexity in the catatonia group in a single cluster comprising predominantly the right insula and contiguous brain regions. There were no significant differences between the LZM and ECT sub-groups. Structural analyses The resampled and smoothed cortical surface complexity files (see supplementary materials for details on processing) were entered into a general linear model framework. We tested the hypotheses whether the mean cortical surface complexity at each vertex was statistically different between the catatonia and the patient samples, and between the LZM and ECT subgroups. We used the threshold free cluster enhancement (TFCE) method 16as implemented in the TFCE toolbox (http://dbm.neuro.uni-jena.de/tfce; version 211) using default settings (5000 permutations) for estimating statistics and p-values. For the VBM analyses, we used the smoothed, modulated, normalized grey matter images with an absolute threshold masking of 0.1 and included the total intracranial volume as a covariate of no interest. We tested the same set of hypotheses as the cortical surface complexity analyses using the same TFCE approach. Reliability analyses In order to estimate the generalizability of our results to the population, we performed reliability analyses of the between-group comparisons that tested our hypotheses (whole-brain and within-network rs-FC), using an iterative jackknife approach. 17We repeated these analyses by iteratively leaving out the data of one subject each from both catatonia and healthy groups. This gives us an estimate of the reliability of the results due to perturbations in the sample (see Supplementary material for details). Data availability The data used in this study is available from the corresponding author, upon a reasonable request. Results We observed widespread increased connectivity in the catatonia sample in the long-range connections from the sensorimotor, salience, frontoparietal, temporal, and cerebellar regions, while hypoconnectivity was noted in the regional and/or inter-hemispheric connections of the sensorimotor, temporo-parietal and cerebellar regions (Fig. 1, Supplementary Fig. 2, and Supplementary Table 7). The LZM subgroup showed significantly higher whole-brain connectivity between several pairs of regions, as compared to the ECT subgroup (Fig. 1, Supplementary Fig. 5 and Supplementary Table 9); most of these connections were from the left paracingulate region. The rs-FC results showed fair reliability (Dice coefficient: 0.60 ± 0.08), with eight pairs of connections showing consistent significant between-group differences across all jack-knife samples (see Supplementary material). Within-network connectivity analysis revealed reduced connectivity in the catatonia sample in sensorimotor, salience, frontoparietal, and cerebellar networks (Fig. 1, Supplementary Fig. 6 and 7, and Supplementary Table 10), with no significant findings within the subcortical network or between the LZM and ECT subgroups. The Dice coefficients for reliability analysis of within-network functional connectivity differences between catatonia and healthy samples were ‘perfect’ 17for the sensorimotor (1.00 ± 0.00), ‘high’ 17for the frontoparietal (0.73 ± 0.12), and fair for the salience (0.63 ± 0.07) and cerebellar (0.62 ± 0.08) networks. All connections in the sensorimotor network, and multiple connections in the salience, frontoparietal and cerebellar networks were consistently different between the groups across all jack-knife samples. The sensorimotor network retained an excellent Dice coefficient (0.92 ± 0.15) indicating high reliability even after an additional Bonferroni correction for the five networks (Supplementary Fig. 8). Left PCG seed-to-voxel functional connectivity analysis revealed four clusters of increased connectivity in the catatonia sample covering the frontoparietal and cerebellar regions (Fig. 1, Supplementary Fig. 10 and Supplementary Table 12). Similar to the whole brain connectivity results, significantly higher seed-to-voxel connectivity was seen in the LZM group covering the frontal and occipital regions (Fig. 1, Supplementary Fig. 12 and Supplementary Table 14). The left PCG seed-based connectivity showed a direct positive relationship with BFCRS motor sub-score (cluster size: 35 voxels; size p-FWE = 0.06, size p-FDR = 0.03; Supplementary Fig. 13). We found a cluster of reduced vertex-wise cortical complexity in the catatonia sample in the right insular cortex and contiguous areas ( p< 0.05 FWE corrected, TFCE, p= 0.01, 272 vertices in size) (Fig. 1, Supplementary Fig. 14). We did not find any volumetric differences in the VBM analysis (TFCE, p< 0.05, FWE corrected). Additionally, we did not find any differences between the LZM and ECT subgroups in VBM or cortical complexity analyses at this threshold. Discussion While the ‘psychomotor refractory state’ hypothesis of catatonia needs further validation, it provides a framework for understanding how benzodiazepines, somewhat paradoxically, produce marked clinical improvement in patients with acute retarded catatonia. In this initial study, we find preliminary evidence for how this aberrant hyperconnectivity is more pronounced in the lorazepam responder group in comparison to the lorazepam non-responder group. These novel findings have potential clinical applications in emergency settings to predict response to benzodiazepines and identify those patients who require early initiation of ECTs for recovery from acute retarded catatonia. Furthermore, this demonstration of the link between neuroimaging markers and treatment response in acute retarded catatonia may pave the way for similar functional neuroimaging studies, including pharmaco-imaging studies in other psychiatric disorders as well, which will potentially enhance our understanding of disease neurobiology and treatment. Data Availability The data used in this study is available from the corresponding author, upon a reasonable request. Funding P.P. acknowledges salary support from the Accelerator Program for Discovery in Brain Disorders using Stem Cells (ADBS) project, funded by the Department of Biotechnology, Government of India (BT/PR17316/MED/31/326/2015). No specific funding was received towards this work. Competing interests The authors report no competing interests. Supplementary material Supplementary material is available online. Author contributions The author contributions are based on Contributor Roles Taxonomy (CRediT) (https://casrai.org/credit/): PP: Methodology, Software, Validation, Formal analysis, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization AG:Conceptualization, Investigation, Data Curation, Writing – Review & Editing VSKR:Conceptualization, Resources, Writing – Review & Editing, Supervision JS:Conceptualization, Investigation, Resources, Writing – Review & Editing, Supervision, Project administration JPJ:Conceptualization, Validation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision, Project administration Acknowledgements We acknowledge the support from NIMHANS for carrying out this research as part of the M.D. dissertation of A.G. We thank Mr. Krishnendu Vyas for assistance with the data acquisition of healthy subjects. Abbreviations BFCRS Bush Francis Catatonia Rating Scale BOLD blood oxygenation level-dependent DICOM Digital Imaging and Communications in Medicine ECT electroconvulsive therapy EPI echo planar imaging fMRI functional MRI HRF hemodynamic response function HS healthy subjects LZM lorazepam NIfTI The Neuroimaging Informatics Technology Initiative NIMHANS National Institute of Mental Health and Neurosciences PCG precentral gyrus pHFO pathological high frequency oscillations ROI region of interest rs-FC resting state functional connectivity TFCE threshold free cluster enhancement VBM voxel-based morphometry References 1. ↵ Fink M. Rediscovering catatonia: the biography of a treatable syndrome . Acta Psychiatrica Scandinavica . 2013 ; 127 ( 441 ): 1 – 47 . doi: 10.1111/acps.12038 2. ↵ Haroche A , Rogers J , Plaze M , et al. 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National Center for Biotechnology Information (US); 2012 . https://www.ncbi.nlm.nih.gov/books/NBK98191/ 20. ↵ Liu S , Parvizi J. Cognitive refractory state caused by spontaneous epileptic high-frequency oscillations in the human brain . Science Translational Medicine . 2019 ; 11 ( 514 ). doi: 10.1126/scitranslmed.aax7830 21. ↵ Ungvari GS , Leung CM , Wong MK , Lau J. Benzodiazepines in the treatment of catatonic syndrome . Acta Psychiatrica Scandinavica . 1994 ; 89 ( 4 ): 285 – 288 . doi: https://doi.org/10.1111/j.1600-0447.1994.tb01515.x Yassa R , Iskandar H , Lalinec M , Cleto L. Lorazepam as an Adjunct in the Treatment of Catatonic States: An Open Clinical Trial . Journal of Clinical Psychopharmacology . 1990 ; 10 ( 1 ): 66 – 67 . 23. ↵ Suchandra HH , Reddi VSK , Aandi Subramaniyam B , Muliyala KP . Revisiting lorazepam challenge test: Clinical response with dose variations and utility for catatonia in a psychiatric emergency setting . Aust N Z J Psychiatry. Published online October 30, 2020:0004867420968915 . doi: 10.1177/0004867420968915 24. ↵ Chumbley JR , Friston KJ . False discovery rate revisited: FDR and topological inference using Gaussian random fields . Neuroimage . 2009 ; 44 ( 1 ): 62 – 70 . doi: 10.1016/j.neuroimage.2008.05.021 The fMRI signature of acute catatonic state and its response to benzodiazepines Pravesh Parekh , Anirban Gozi , Venkata Senthil Kumar Reddi , Jitender Saini , John P. John medRxiv 2021.03.23.21253765; doi: https://doi.org/10.1101/2021.03.23.21253765	https://www.medrxiv.org/content/10.1101/2021.03.23.21253765v1.full
Universities have gone from being a place of privilege to a competitive market. What will they be after coronavirus? \| Evening Report Academic Analysis Analysis Features MIL-OSI Universities have gone from being a place of privilege to a competitive market. What will they be after coronavirus? Source: The Conversation (Au and NZ)– By Tamson Pietsch, Associate Professor, Social & Political Sciences, University of Technology Sydney This essay is based on the first episode of the new UTS podcast series “ The New Social Contract ” that examines how the relationship between universities, the state and the public might be reshaped as we live through this global pandemic. Younger Australians will bear the economic, social and environmental costs that come from the COVID-19 pandemic. They’re making sacrifices in the name of public health and to protect the old and vulnerable. The heavy lifting of rebuilding will also fall disproportionately to them. Prime Minister Scott Morrison referred to these sacrifices as “a social contract”. A social contract in this sense is not something that gets recorded in legislation, but more of an unstated agreement that comes from practice, policy and circumstance. It is a reciprocal relationship, in which obligation and benefits rest on all parties – though these aren’t always evenly distributed. How Australia will fare in a post pandemic world depends on this relationship between institutions, society and the state. And universities have a crucial role to play, by providing public goods such as understanding, training and research. The social contract for universities has changed several times. Its different versions can be seen in the decisions students have had to make on entering university at different points across the 20th century. These decisions tell us a lot about how the social contract for universities in Australia has changed, and what might be possible as it changes again. Early 1900s – the family In 1910 a student, let’s call him Frederick, had his family at the centre of decision-making. Frederick is from Bendigo where his father is a shopkeeper. He is eager to study Medicine at the University of Melbourne. But before he even sets foot on campus, he has to sort out his finances. That he can even attend university is thanks to his uncle Jim, a successful doctor. His uncle is paying for Fred’s textbooks, microscope, accommodation, living expenses and tuition fees – though the latter are only a minor component of the costs. Fred and his uncle both expect the university will provide an education on par with that offered in British universities – training that will enable Fred to attain the cultural capital necessary for middle-class society and the technical knowledge to practise as a doctor. On graduation, he knows he is expected to make his uncle proud by returning to Bendigo and joining the family practice. Frederick’s story reflects the broad terms of a social contract under which universities, which received abouthalf of their fundingfrom state governments, trained a relatively small cohort of professionals. In return for social status, these professionals provided expert services to a rapidly growing society. State government annual grants supported universities as institutions that would build the society and economy of the new Australian nation. The 1910 student has family at the forefront of his decision-making. (Students Ormond College, Melbourne University 1896/1902) James Fox Barnard/State Library Victoria Research played a minor role,with some funding from private companies and state governments. As far as the student was concerned, the cost (or risk) of attending university was borne by the family (or other patron) and it was to the family obligations were owed. Mid-20th century – employers Forty years later, Margaret is one of a growing number of women entering university. On her first day in a teaching degree at the University of Queensland, she meets chemistry student Eric. They find they have a lot in common. They have both joined UQ’s chapter of the Student Christian Movement, and have already signed contracts with their future employers who will financially support them during their studies. The Queensland education department has awarded Margaret a bonded scholarship based on her high school matriculation results. Eric has taken a cadetship with the CSIRO. After they graduate, both will have to work on their employers’ terms for three to five years. Margaret’s and Eric’s stories reflect the terms of a social contract that emerged in the middle of the 20th century. Under this model, the costs of higher education could be borne by a student’s future employer such as government departments like the Postmaster-General or the railways, or private entities such as manufacturers and mining companies. Connected to an expanding network of state secondary schools, this new pathway delivered benefits to students who would not otherwise have been able to go to university. This enabled them to join the ranks of the expanding white collar, salaried middle class, in return for a commitment to work. The same logic underpinned the creation of two postwarCommonwealth programs: the Commonwealth Reconstruction Training Scheme, which enabled 21,000 ex-servicemen and women to receive a free tertiary education, and Commonwealth Scholarships, which covered fees and in some cases living expenses on the basis of secondary-school results. Employers benefited, but so did the state, which saw this as a more directed way of providing funding to universities to produce skilled graduates in needed areas. Eager to boost post-war development and in the strategic context of the Cold War, universities expanded research, helped by new direct grants from the Commonwealth. 1970s – society It’s 1975 and Daryl, having passed his high school matriculation exam, enrols in therecently opened Macquarie Universitywithout having to worry about finances at all. Since 1974,fees have been abolishedand, once Daryl fills out the right form, he can get student assistance for living expenses based on a means test rather than a competitive exam. Like increasing numbers of his generation, Daryl decides to study science. But he can switch degrees if he finds he is better suited to another program. He lives in a share-house with other students. With less pressure to pass every subject to keep his place, he enjoys his social life in an inner suburb. He forms a punk rock band with some university friends and, for a few years after graduation, tours pubs around Australia. In the mid-20th century, the costs of higher education were often borne by the student’s employer. (First Students at Macquarie University, Sydney. 1967) Wikimedia Commons Eventually Daryl settles into a job at the Australian Bureau of Statistics. He feels lucky to find employment in an expanding public sector during a period of economic stagnation. The agreement Daryl made on entering university was not with his family or future employer, but with the state itself, when he applied for student assistance. Prime Minister Gough Whitlam’s government abolished fees andreduced financial barriers to study. This reallocated the cost of higher education from state governments to the Commonwealth, and the benefit to society. Daryl benefited because he got a secure job in the public service. And society benefited through his skilled employment and active participation in the cultural and social life of the nation. The Commonwealth also funded research to support economic development, productivity and defence. 1990s – the individual No one in Ashley’s family has ever been to university before. But the introduction of the newHigher Education Contribution Scheme(which means she doesn’t have to pay fees until she begins earning money) has opened places for people like her. Conscious HECS is just a loan, Ashley wants a degree that gets her a job. That’s why she picks Communications. And because she’s interested in video production, she gets involved in the film society. Ashley hopes this might give her an advantage in a very competitive industry. She doesn’t have much time for socialising, because she also has a part-time job to help pay her living expenses. Ashley’s experience reflects a social contract ushered in during the late 1980s and early 1990s. With the reintroduction of student fees and creation of HECS, students knew they were paying a much larger contribution to their higher education. They expected direct benefits in terms of future employment options and income. An emphasis on “human capital” came to the fore and universities were told to equip students with skills they could can take into the job market. COVID-19 has pulled on the threads of the already worn fabric of higher education policy. (University funding cuts protest, 2017) Callum Godde/AAP They also competed for a reduced amount of research dollars which were now distributed on the basis of competitive application through a newly created entity,the Australian Research Council. And they competed with high paying international students whosenumbers grew by 2,000%between 1986 and 2006, providing an extra non-government source of income. Competition created national and global university rankings and research metrics as a way of measuring value. This social contract worked within the terms of the market economy. 2020 – a new social contract? While on average,graduates earn more than non-graduates, a degree no longer guarantees employment. Going to university is more expensive that before, and its returns are less guaranteed to convert into personal benefits. And COVID-19 has pulled on the threads of the already worn fabric of higher education policy. The deferred nature of the HECS payment, use of market mechanisms to allocate value, and the enormous supplement that comes from international student fees, has pushed the idea of the social contract for higher education out of view. After a summer of devastating fire,universities,societyandeven some Australian stateshave recognised the country needs a social and economic framework dedicated to the conditions of habitability. To achieve that, care for the planet, and each other, must be at the heart of all we do. COVID-19 has also revealed people’s willingness to participate in collective action is just as crucial to effecting transformation as is expertise. What does that mean for universities? What is their purpose in the 21st century? What new set of obligations and expectations will students face? What should we ask of them? What role should government play? These are the questions our sector should be asking as we face lengthening months and years in which the world higher education in Australia, and the lives of all those who rely on it, is likely to grow even more precarious. Next week’s podcast will explore the current context of the crisis for the university sector. Universities and the public in the 20th Century was made by Impact Studios at the University of Technology, Sydney – an audio production house combining academic research and audio storytelling. – ref. Universities have gone from being a place of privilege to a competitive market. What will they be after coronavirus? – https://theconversation.com/universities-have-gone-from-being-a-place-of-privilege-to-a-competitive-market-what-will-they-be-after-coronavirus-137877	https://eveningreport.nz/2020/05/06/universities-have-gone-from-being-a-place-of-privilege-to-a-competitive-market-what-will-they-be-after-coronavirus-137877/
A Short Credo on Justification: Douglas Wilson \| Page 3 \| The Puritan Board Originally posted by WrittenFromUtopia Joseph, you are confounding Justification and Sanctification, in my opinion, when you say " A faith that does not... A Short Credo on Justification: Douglas Wilson Originally posted by WrittenFromUtopia Joseph, you are confounding Justification and Sanctification, in my opinion, when you say "A faith that does not work is a faith that does not save." Gabriel, thank you for your input. I certainly don't want to be argumentive . . . I honestly want to understand your perspective . . . please explain to me how it is that you think my statement confounds justification with sanctification? I do not understand why you are making that charge. I believe (as did all the Reformers) that "a faith that does not work is a faith that does not save." Please help me understand how that statement is any different whatsoever from these sample statements: As Dr. John Gerstner said : "Faith is Not a Work, but it is Never without Work" As Brian Schwertly said : "Justification is by faith alone, but not by the faith that is alone. Biblical Protestants agree with the apostle James "œthat faith without works is dead" ( Jas. 2:20 ). " "œJustification is by faith alone, but not by the faith that IS alone." - Martin Luther Like I said: "A faith that does not work is a faith that does not save." Now, let me break up that statement into its constituent parts. First of all, I am saying that there are two different kinds of faith. There is true faith, and there is dead faith. Do you agree with me so far, Gabriel? If so, great. (If not, then let's stop here and see why we disagree on this point.) True faith is self-explanitory. It is simple honest belief in Jesus Christ for the salvation from sin. This is real faith. Dead faith is false faith. It may outwardly appear identical to true faith for a while . But from day one, it never truly trusts in Christ for salvation. Thus, it is not real faith at all. Perhaps this could be better termed "apparent faith". Now let me ask you: Which one of these two kinds of faith can save? Does God justify people on the basis of true faith, on the basis of dead faith, or on the basis of both? I would argue from Scripture that ONLY true faith is salvific. Dead faith cannot save (James 2 ). Apparent faith is not good enough ( Matthew 7:21-23 ). In other words, I am saying that salvation is only granted to true, actual, real faith. If a person honestly believes in Christ for salvation, then he is justified by faith alone. Works have absolutely NOTHING to do with this imputed righteousness received by the sinner. ** Now, on a totally SEPARATE note *** Of the two types of faith, which one produces good fruit? Clearly, the dead faith does not produce good fruit. The true faith, though, most certainly WILL produce good works. As you well pointed out, this is sanctification, NOT justification. This is progression in holiness, NOT imputed righteousness. Now let's ask the question: Q: "Which faith saves?" Answer: "Only True faith saves. Dead faith does not save." Now let's ask this: Q: "Which faith works?" Answer: "Only True faith works. Dead faith does not bear good fruit." ------------- We have made two separate and distinct statements. Now let's succinctly say BOTH distinct truths in a single statement --------- "A faith that does not work is a faith that does not save." This statement is perfectly logical, and is perfectly TRUE. Notice what is NOT said in the statement. The statement does NOT say that the salvation is because of the works! If it said THAT, then I WOULD be confounding justification with sanctification. But I am saying nothing of the sort. Rather, I am simply pointing out that there are two different kinds of faith, and that only one kind of faith saves. True faith saves; dead faith does not save. True faith works; dead faith does not work. What kind of faith is true faith? It is a faith that results in works! Therefore, what kind of faith saves? The only faith that saves is the faith that works! But it does not save BECAUSE it works. Rather, salvation & works are two DIFFERENT things that EACH flow out from true faith SEPARATELY. In other words, true faith results in both justification AND sanctification. The justification is granted BECAUSE of the faith. The justification is NOT granted because of the works. Roman Catholic theology looks like this: Reformed theology looks like this: Justification comes on the basis of true faith. Works flow out of true faith. Thus, "the faith that works is the faith that justifies". And since dead faith brings about neither justification nor good works, we can also say that "the faith that doesn't work is the faith that doesn't justify". The negatives always go together, and the postives always go together. (It is kind of like people in the Bible sparing someone's life, judging by how they pronounce the word "Shibboleth". They did not save someone or kill someone because they wanted the word pronounced a certain way. Rather, the basis for saving or killing a person was based on totally different grounds. But they just used their pronunciations of the word "Shibboleth" as a way to properly identify the people they were looking for. It was a method of identification , NOT a grounds for saving or killing someone.) As you can clearly see in the diagram, justification is NOT on the basis of works, and does NOT flow from works in any way. Justification is on the basis of faith ONLY. Justification and works both come from faith, but only side by side, in parallel lines. Make sense? If I may as an observer. It is clear to me, I think, that Joseph is only articulating the reformed position. The original discussion is whether Wilson agrees with this position. Based on what has been stated I think he does, if his statements on justification quoted by Joseph are left alone. However as Sean and others have pointed out, it is the other stuff (faith, works, covenant, etc.) that seems to take away any area of orthodoxy on justification that was stated. To be clear, I am not a Wilson advocate, and I am not pro-FV, however I don't think it is fair to Joseph to paint him in to a corner that he is clearly articulating he wants no part of. If Joseph is confused, he is confused about Wilson agreeing with the truth. I don't think he is confused about what that truth is. I may be wrong, but I have no horse in this race and am giving Joseph the benefit of the doubt. That being said, I think the discussion would progress better if a focus on the definition/nature of faith would be given by Joseph and Sean. If faith is reduced to mental assent, than Joseph's point is confirmed by historic reformed orthodoxy. If faith is revised to include works as part of the nature of faith itself as opposed to a fruit of that faith, then Sean's point is confirmed by historic reformed orthodoxy. Anyways, just thought I'd feebly attempt to spur on the discussion towards clarity. Maybe discussing the nature of faith would help? [Edited on 4-7-2006 by RAS] Originally posted by RAS If I may as an observer. It is clear to me, I think, that Joseph is only articulating the reformed position. Thank you! Originally posted by RAS The original discussion is whether Wilson agrees with this position. Correct. And I am arguing that Wilson believes the reformed position every bit as much as I do. Originally posted by RAS Based on what has been stated I think he does, if his statements on justification quoted by Joseph are left alone. Again, thank you! Originally posted by RAS However as Sean and others have pointed out, it is the other stuff (faith, works, covenant, etc.) that seems to take away any area of orthodoxy. Please clarify what you mean by this statement. Specifically what is it that Wilson has said about "faith, works, covenant, etc." that causes you to question his orthodoxy in any way? I have already discussed Wilson's statements about faith & works, and they are no different from the classic reformed view of faith and works. The faith that works is the same faith that saves. But the salvation is based only on the faith, not on the works. So what is wrong with Wilson's view, in your opinion? Originally posted by RAS That being said, I think the discussion would progress better if a focus on the definition/nature of faith would be given by Joseph and Sean. If faith is reduced to mental assent, than Joseph's point is confirmed by historic reformed orthodoxy. To have true faith, you have to: 1) have knowledge (content) 2) mental assent to that knowledge (conviction) 3) trust in Christ according to that conviction (personal confidence) Works have nothing to do with it. True faith ALWAYS results in salvation. True faith ALWAYS results in good works. Thus, the faith that works is the same faith that saves. But the salvation is based only on the faith, not on the works: Originally posted by RAS If faith is revised to include works as part of the nature of faith itself as opposed to a fruit of that faith, then Sean's point is confirmed by historic reformed orthodoxy. Wilson (and the rest of the reformed community) say that good works are the fruit of true faith. Wilson (and the rest of the reformed community) do not revise faith to include works as part of the nature of faith itself. For example, apples are not a tree, and a tree is not an apple. The nature of "apple treeness" does not include apples in its definition. An apple tree can live for years as a sapling without ever bearing apples, and yet it still is an apple tree. However, it is the nature of the apple tree to bear fruit eventually. It is coded into the DNA of the tree itself. The bearing of fruit is NOT what makes it an apple tree, but rather is the natural outgrowth of what that tree is in and of itself, apart from its fruit. --- The nature of faith does not include works as part of its definition. Nevertheless, the nature of faith is such that it eventually WILL bear good fruit. Good works are not part of the definition of faith, but the proclivity toward good works is built into the "DNA" of faith itself. A true faith saves. And true faith will eventually bear fruit. It is within its nature to do so. Now, good works do not just "maybe" flow from true faith. Rather, good works definitely flow from true faith. Thus, it is a true statement that faith and works are inseparable . However, faith and works are not identical . Wilson DOES say faith and works are inseparably tied together, as has said every other Reformed theologian in history. A faith that doesn't work is not true faith at all, because true faith always bears the fruit of good works. Wilson does NOT say that faith and works are the same thing. Originally posted by RAS Anyways, just thought I'd feebly attempt to spur on the discussion towards clarity. Maybe discussing the nature of faith would help? (back to the sidelines...) THANK YOU very much for your contribution to this thread! I agree that the definition of faith is very important. Ironically, I think the problem is that many opponents of Wilson are the ones who don't engage this argument with enough precision. Too many people mistake the word "inseparable" for "identical", and thereby mistake Protestant theology for Roman Catholic theology. If faith and works are one and the same, then we are talking Roman Catholicism. If faith and works are inseparably tied to one another, then we are talking Protestantism. Works do not save. Faith and works are not the same thing. True faith always works. True faith always results in salvation. The faith that works is the same faith that saves. Justification is by faith alone, but not by the faith that IS alone. Sean, thanks for clarifying. I am glad to know that I did understand you. One more question. Assuming you allow the existence of a dead faith, what distinguishes dead faith from living faith? Of course I allow for the existence of a dead faith, but unlike Wilson who argues that a dead faith is no different from true faith with the exception that it lacks works, I would say a dead faith is no faith at all. The term "œdead faith" is a metaphor. A word picture. Dead faith means lip service -- a feigned faith; the faith of a hypocrite. It is a person who says he believes, but really doesn´t. Who claims to assent to the truth of the Gospel, but does not. Remember, per Wilson, hypocrites are Christians and by their baptism they are the recipients of ALL the benefits of the Covenant (except obviously for the gift of faith and salvation). Amazingly and according to Wilson, "œboth the true and false son are brought into the same relation." Wilson doesn´t understand that the phrase "faithless Christian" is a contradiction in terms, and a "nominal Christian" is a person who acts like, but is not, a Christian -- the sort of hypocritical church member James discusses in James 2 . Wilson denial in his book that there are "nominal Christian" implies that all hypocrites are Christians. But the Bible speaks of "false brethren," "false teachers," and "false prophets," all of whom are nominal Christians. To restate: for Wilson a person CAN believe the Gospel, assent to the truths of Scripture and Christ´s finished work on their behalf and still be lost. Like those who will one day cry "œLord, Lord" and point to their many outstanding works as evidence of their faith, and of whom Jesus will turn and say "œI never knew you," Wilson also would have us look to our works as evidence of our faith in order to receive our "œfinal justification." Therefore it follows, that belief alone is not enough to justify a man before the bar of divine justice. I would ask Joseph, as Wilson´s able defender and advocate on these boards (or at least the only one so far with the courage to show his face ), would he "“ or would you, since you´re asking the question -- agree with the following : . . . James does not see anything wrong with the faith he is talking about. The faith isn´t the problem; the fact it is alone is the problem. To understand what kind of faith James has in mind, one must avoid the temptation to read something bad into it. This is where the "mere intellectual assent" solution went wrong. Its advocates correctly identified verse 19 as the key to understanding the faith being discussed, which is intellectual assent. The problems were created by adding the term "mere" to make it sound bad. Leave "mere" off, and the problems vanish. Someone can go around boasting that he intellectually assents to God´s truth (v. 14), prompting James´s need to show that intellectual assent without works is dead and barren (vv. 17, 20, 26). He could offer to show his intellectual assent by his works (v. 18). And he could commend a person for having intellectual assent (v. 19a), while saying that even the demons have it though it doesn´t stop them from shuddering at the prospect of God´s wrath (v. 19b). Finally, he can speak of how Abraham´s intellectual assent was active with and completed by his works (v. 22) and can conclude that man is not justified by intellectual assent alone (v. 24). James views intellectual assent as good thing ("you do well," v. 19a), but not as a thing that will save us by itself (vv. 14, 17, 20, 24, 26). You also asked: For Wilson, from your quoting of him, it seems to be that one is assent and another is assent plus something. How would you distinguish the demonic belief from the believer's belief? Let´s make sure we first agree with what Wilson is saying and that the assent plus something else which makes faith saving is an assent plus works; "œThe works of which he is speaking are works that spring from faith and are the expression of faith . . . Without them [works done by faith] no man can inherit the kingdom of God ( Gal. 5:21 ). " Do we agree? Just an aside, it´s also important to note that Gal. 5:21 does not say anything at all what Wilson claims. What the verse states is; "œ. . . envying, drunkenness, carousing, and things like these, of which I forewarn you just as I have forewarned you that those who practice such things shall not inherit the kingdom of God." Wilson´s inference is invalid. The verse does not say nor does it imply that works done by faith are necessary in order to inherit the kingdom of God. Wilson doesn´t understand the alien righteousness of Christ; the righteousness of faith. In his sensate and "œphotographical" religion Wilson can only understand what he can see with the eyes in his head and that´s pretty doubtful too. Anyway, I just want to make sure we´re on the same page before we can discus demonic theology, which is, after all, what I thought we´ve been discussing all along. That being said, I think the discussion would progress better if a focus on the definition/nature of faith would be given by Joseph and Sean. I'm in agreement with Gordon Clark who defines faith as an assent to understood propositions. I think the addition of trust or "œfiducia" as a third component which is supposed to make faith "œsaving" is to, in essence, define the word faith with itself (since one of the synonym for belief per my thesaurus is trust) and adds nothing to the definition or to our understanding of what faith is. I would strongly recommend anyone interested to read Clark´s "œWhat is Saving Faith," even if only to see the profound confusion that has been the result of adding this third and mostly undefined element which is supposed to make ordinary belief salvific. OTOH, my hat is off to Wilson who is very clear in what he means by "œfiducia," even to the destruction of the Gospel. . . . James does not see anything wrong with the faith he is talking about. The faith isn´t the problem; the fact it is alone is the problem. To understand what kind of faith James has in mind, one must avoid the temptation to read something bad into it. This is where the "mere intellectual assent" solution went wrong. Its advocates correctly identified verse 19 as the key to understanding the faith being discussed, which is intellectual assent. The problems were created by adding the term "mere" to make it sound bad. Leave "mere" off, and the problems vanish. Someone can go around boasting that he intellectually assents to God´s truth (v. 14), prompting James´s need to show that intellectual assent without works is dead and barren (vv. 17, 20, 26). He could offer to show his intellectual assent by his works (v. 18). And he could commend a person for having intellectual assent (v. 19a), while saying that even the demons have it though it doesn´t stop them from shuddering at the prospect of God´s wrath (v. 19b). Finally, he can speak of how Abraham´s intellectual assent was active with and completed by his works (v. 22) and can conclude that man is not justified by intellectual assent alone (v. 24). James views intellectual assent as good thing ("you do well," v. 19a), but not as a thing that will save us by itself (vv. 14, 17, 20, 24, 26). Sean, Who wrote this quote? Originally posted by Dan.... Who wrote this quote? Catholic Answers: http://www.catholic.com/thisrock/1997/9710chap.asp Thats funny. That quote is from James, an old friend of mine. I havent seen him in a few years. I've been meaning to get back in touch with him. He is VERY into trying to convert protestants into Catholics. He is very well studied. He definately drove me to search the scriptures. (Sorry for getting off subject.) Of course I allow for the existence of a dead faith, but unlike Wilson who argues that a dead faith is no different from true faith with the exception that it lacks works, I would say a dead faith is no faith at all. The term "œdead faith" is a metaphor. A word picture. Dead faith means lip service -- a feigned faith; the faith of a hypocrite. It is a person who says he believes, but really doesn´t. Who claims to assent to the truth of the Gospel, but does not. So dead faith is merely the insincere profession of a hypocrite, or the sincere but deluded profession of a self-deceived person? How would that then tie in to James' statement that the devils believe? I ask again, knowing that you asked me to answer another question first, viz, this one: Let´s make sure we first agree with what Wilson is saying and that the assent plus something else which makes faith saving is an assent plus works; "œThe works of which he is speaking are works that spring from faith and are the expression of faith . . . Without them [works done by faith] no man can inherit the kingdom of God ( Gal. 5:21 ). " Do we agree? I am not sure if we agree, because I am not sure I understand you --and clarifying in what sense the demons can be said to believe would help me to do so, I think. I gathered from your statement above that Wilson's view is this: Assent plus works is saving . Joseph, of course, believes that you are wrong. He thinks, if I understand him correctly, that Wilson is saying this: Saving faith always produces works . Of course, you and Wilson disagree on the question of whether dead faith is merely professed faith without true belief, or whether it is intellectual assent simpliciter While I'm at it, I would like to ask Joseph again, as it kind of got buried in a flurry of posts. Between the statements that, A. justification is on the basis of faith or B. justification is through faith which would you choose? [Edited on 4-8-2006 by py3ak] So dead faith is merely the insincere profession of a hypocrite, or the sincere but deluded profession of a self-deceived person? I don´t see that it matters, in both cases the profession is a false one. How would that then tie in to James' statement that the devils believe? This is what Calvin says: "œ. . . it would be ridiculous were any one to say, that the devils have faith; and James prefers them in this respect to hypocrites."œ Can you guess what Calvin would say about Wilson? : Let´s make sure we first agree with what Wilson is saying and that the assent plus something else which makes faith saving is an assent plus works; "œThe works of which he is speaking are works that spring from faith and are the expression of faith . . . Without them [worksdone by faith] no man can inherit the kingdom of God ( Gal. 5:21 ). " Do we agree? I am not sure if we agree, because I am not sure I understand you --and clarifying in what sense the demons can be said to believe would help me to do so, I think. I gathered from your statement above that Wilson's view is this: Assent plus works is saving. Joseph, of course, believes that you are wrong. He thinks, if I understand him correctly, that Wilson is saying this: Saving faith always produces works. Of course, you and Wilson disagree on the question of whether dead faith is merely professed faith without true belief, or whether it is intellectual assent simpliciter Well, Ruben, it´s hard for me to see any qualitative difference between the argument against justification by faith alone in the quote I provided from Catholic Answers and in the selection I provided from Wilson´s blog. Also, there is nothing "œmere" about assent and in Wilson´s case its addition is an unnecessary rhetorical device. Wilson doesn´t understand what´s entailed in assent and he evidently can get a lot of milage out of the ignorance of many. If you can find any substantial disagreement between the argument presented by these men, let me know? Look at them again: Wilson : "œThe demons also, he says, have that sort of faith, and yet evidently they are not saved ( James 2:19 )." Catholic Answers : Someone can go around boasting that he intellectually assents to God´s truth (v. 14), prompting James´s need to show that intellectual assent without works is dead and barren (vv. 17, 20, 26). He could offer to show his intellectual assent by his works (v. 18). And he could commend a person for having intellectual assent (v. 19a), while saying that even the demons have it though it doesn´t stop them from shuddering at the prospect of God´s wrath (v. 19b). Wilson : "œThe answer is perfectly plain. The faith which James is condemning is a mere intellectual assent which has no effect upon conduct." Catholic Answers : James does not see anything wrong with the faith he is talking about. The faith isn´t the problem; the fact it is alone is the problem. Wilson : "œWhat Paul means by faith . . . is not . . . intellectual assent to certain propositions, but an attitude of the entire man by which the whole life is entrusted to Christ . . . Faith working through love" is the key to an understanding both of Paul and James. The faith about which Paul has been speaking is not the idle faith which James condemns, but a faith that works. Catholic Answers : "œFinally, he can speak of how Abraham´s intellectual assent was active with and completed by his works (v. 22) and can conclude that man is not justified by intellectual assent alone (v. 24)." Both Wilson and Rome deny that belief alone in the understood propositions of the Gospel are enough to save a man or is what makes faith "œsaving." Even if we disregard "œassent simpliciter" everyone should see that both assert that the addition of works done by faith are what make faith "œcomplete" or saving. So, for our purposes here, I could care less what Joseph thinks concerning what Wilson has written. If you can´t see that Wilson and Rome are in agreement concerning James and the nature of saving faith, then I frankly can´t see how I would have any better chance in answering your questions then I was in addressing Joseph´s many objections. I will just be wasting my time and yours. However, since so many seem so patently blind to the thinly veiled false gospel promulgated by Wilson and his allies, it gives me even less hope that God will cause men to rise up and throw these base heretics out of our ranks. Actually, if they would all leave and join Wilson´s denomination that would be a great blessing, but of course they won´t. I think perhaps the only solution is the one offered by Paul Elliot in his book, Christianity and Neo-Liberalism, which can be summed up; "œcome out from among them, and be ye separate, saith the Lord . . . ." Sean, Thanks again for your reply. I feel like I am slowly making some headway in understanding where different people are coming from on this whole issue. I grant you that Catholic Answers and Wilson sound very similar. But I wonder if there isn't a subtle difference at this point? Even if we disregard "œassent simpliciter" everyone should see that both assert that the addition of works done by faith are what make faith "œcomplete" or saving. With Catholic Answers that does seem extremely clear. With Wilson's statements it seems possible that there is still one other possibility. According to Catholic Answers it is the addition of works to faith that makes faith saving. I think Wilson may be saying that is the character of saving faith to work. Obviously, they agree in taking "faith" in James 2 to refer to assent. But in one case the point would be: without works faith is not saving, because works are essential to justification , whereas in the other case it would be: without works faith is not saving, because genuine faith is a living, busy active faith, and the lack of works demonstrates the falsity of the faith, without the works being in any sense the grounds or instrument of justification . Does that make any sense? I have been ignorant throughout most of this discussion . . . ignorant about Sean Gerety, that is. I finally realized that Sean co-wrote a book with John Robbins. No wonder this discussion is going nowhere! I will still respond to some of the posts above, as I have time, just because other people are reading this thread. But no longer will I attempt to change Sean's mind. If he is comfortable working with John Robbins, then it is a waste of time for me to try to convince him of anything whatsoever. Frankly, I would consider it an HONOR to be anathematized by John Robbins! If John ever bothers to call me a heretic, I will gladly find a link to where he does so, and I will proudly display the link on my website. My opinion is that Robbins is a nut; and I categorically ignore what he has to say. I am amazed that anyone would want to write a book with John Robbins. However, I do hope Sean is better than Robbins. I guess only time will tell. [Edited on 4-8-2006 by biblelighthouse] Originally posted by py3ak Sean, Thanks again for your reply. I feel like I am slowly making some headway in understanding where different people are coming from on this whole issue. I grant you that Catholic Answers and Wilson sound very similar. But I wonder if there isn't a subtle difference at this point? Even if we disregard "œassent simpliciter" everyone should see that both assert that the addition of works done by faith are what make faith "œcomplete" or saving. With Catholic Answers that does seem extremely clear. With Wilson's statements it seems possible that there is still one other possibility. According to Catholic Answers it is the addition of works to faith that makes faith saving. I think Wilson may be saying that is the character of saving faith to work. Obviously, they agree in taking "faith" in James 2 to refer to assent. But in one case the point would be: without works faith is not saving, because works are essential to justification , whereas in the other case it would be: without works faith is not saving, because genuine faith is a living, busy active faith, and the lack of works demonstrates the falsity of the faith, without the works being in any sense the grounds or instrument of justification . Does that make any sense? AMEN, Ruben! You understand it!! Your excellent input definitely makes this thread worthwhile. THANK YOU for taking part in this discussion, Ruben! You have stated Wilson's position PERFECTLY, in my opinion. And you have done an excellent job of demonstrating why Wilson's view is TOTALLY different from Rome's view. A+ superb! Thank you, thank you, thank you!! Your brother in Christ, Joseph Ruben put it better than I did! In a nutshell: "the lack of works demonstrates the falsity of the faith, without the works being in any sense the grounds or instrument of justification." Joseph, An attempt to discredit what has been said regarding Doug Wilson by appealing to a relationship to John Robbins is a genetic fallacy . I realize that many people do not care for Mr. Robbins, but that does not invalidate the issues he raises against FV. Would you make the same arguments against R. Scott Clark, Calvin Beisner, J. Ligon Duncan, et. al. who also see the serious errors of FV? Joseph, I am glad my statements met with your approval. It seems like the only way to make sense of all parts of what Wilson says. An interesting point that came up to me was the difference in the ways of reconciling James and Paul. Some do it by stating that they use 'justification' in a different sense and others by stating that they use 'faith' in a different sense. I think it might be a profitable discussion to hash out that difference (maybe in another thread). What is the evidence for different meanings to justification as opposed to different meanings for faith? It has also caused me to wonder if there are any major splits among Gordon Clark followers? Van Til seems to have left at least two threads; did Clark leave just one, represented by Mr. Robbins? Or is there someone who feels that Robbins does not understand Clark? Well, I just finished reading this whole thread -- I find it interesting that Wilson's Credo on Justification cannot, on its own, settle the matter. Is not a creed written for the express purpose of being clear ? If so, Wilson has failed in this respect . . in my humble opinion. And he certainly manifested a strange twist in it: I believe that God established two distinct covenants with mankind, one before the Fall, and one after. The first covenant was called a covenant of works in the Westminster Confession (7.2). I would prefer to call it a covenant of creational grace. The condition of covenant-keeping in this first covenant was to believe God´s grace, command, warnings, and promise. (Bold = mine) This is strange and novel (do I really have to say that?). The condition had little to do with believing and everything to do with obeying . Well, obeying involves believing, too . . this is true (as the first table of the Commandments makes clear). But, it wasn't merely that he believe, he had to obey! Adam was a recipient of grace, and thus, the sin that plunged our race into death was a revolt against grace. Wilson seems to confuse creation and covenant here. On account of God having created Adam, he had to believe he existed, and he had to obey God with a personal, perpetual, and perfect obedience -- even without the covenant! The goodness of God (note: not the grace of God) is manifest in the "voluntary condescension." The point? Man always owed God obedience, but now God promised to bless that already-owed obedience! This is what expresses the goodness of God. So, Adam would have merited eternal life ex pacto , on account of the covenant. It wasn't that his works were inherently valuable, but that God was pleased, by "voluntary condescension" to attach a promised blessing to that obedience. This is exactly what the Confession says (7.1): The distance between God and the creature is so great, that although reasonable creatures do owe obedience unto him as their Creator, yet they could never have any fruition of him as their blessedness and reward, but by some voluntary condescension on God's part, which he hath been pleased to express by way of covenant. So, to call this a covenant of "creational grace" seems rather silly to me. His definition of this strikingly different covenant from the one the Confession describes. It denies that Adam could have obeyed on his own merits, and seems to confuse grace (prelapsarian/postlapsarian grace?). I am just unclear with what he means by "grace" here. How is God's grace to Adam any different from his grace towards us in Christ? Is Wilson differentiating the two? The second covenant is a covenant of redemptive grace. The thing that the two covenants have in common is grace, not works. No, he's not. That's what makes it confusing. Apparently grace is grace, and the same grace was in the first covenant that manifests itself in the second . . ? "œPerfect and personal obedience," even for an unfallen man, is not possible unless he trusts in God´s goodness and grace. Wilson here seems rather clearly to deny the fourfold state of grace taught explicitly in the Confession's chapter on Free Will. I quote the Confession (9.2): Man, in his state of innocency, had freedom, and power to will and to do that which was good and well pleasing to God; but yet, mutably, so that he might fall from it. For Wilson, apparently Adam didn't have this power? Of course he had to obey God and believe in him, but is such the basis of his power to obey? No, the power to obey was not contingent on his obedience, but on the way God had created him. Strangely, it seems like Wilson is saying "Adam's obedience is based on his obedience." (Yes, in his Credo he quotes from WCF 19.1, but he seems to deny this . . he's trying to ride the fence, it seems.) The condition for keeping this covenant is the same as the first, although the circumstances are different. The condition always is to believe God. Wait a second. If, according to Wilson, grace was required in the Covenant of Works to enable Adam for obedience in order to obey that covenant . . And the conditions of both covenants are the same (obedience, I suppose?), then in the Covenant of Grace is God merely giving the same sort of grace he gave in the Covenant of Works to Adam? Is God merely enabling us to obey? This seems to be what Wilson is saying (but, it's very possible I'm misunderstanding him). These points are made, not to smuggle "œworks" from the covenant of works into the covenant of grace, but rather the opposite. I believe we must insist that autonomous works be banished from every human realm and endeavor, whether fallen or unfallen ( 1 Cor. 1:31 ). Seems to me he's trying to sit on that fence again. He's denying what he just tried to prove. Grace enables Adam for obedience. It's the same grace and same condition in the Covenant of Works as in the Covenant of Redemption. God enables us for obedience, by his grace, in the Covenant of Grace. Am I reading him wrong? (Disclaimer: I have read very little of Wilson's other works, and am merely discussing on the basis of his Credo quoted at the beginning of this thread . . which I supposed should be clear enough to make his point.) Casey, I agree that Wilson seems very confused on the Covenant of Works. Smuggling grace into that covenant seems to be precisely what the Westminster divines were trying not to do. And I think that lack of understanding does play out into the rest of what he says. Of course, I am not a Wilson expert. I recently download Reformed Is Not Enough but so far have found it deeply boring. Originally posted by Jeff_Bartel I realize that many people do not care for Mr. Robbins, but that does not invalidate the issues he raises against FV. Would you make the same arguments against R. Scott Clark, Calvin Beisner, J. Ligon Duncan, et. al. who also see the serious errors of FV? Whereas I do not respect Robbins much, I certainly DO respect Clark, Duncan, etc. However, I think their interpretive errors regarding the FV are just as grave. They miss the point just as badly as Robbins does. McMahon totally misses Wilson's point as well. And I highly respect McMahon! The question keeps coming up: "Could all of these great theologians be wrong?" The plain answer is: YES!! They are all wrong. They do not understand what Wilson is saying. And I do understand what Wilson is saying. If I were to explain Federal Vision theology, FV people would themselves agree that I have respresented them correctly. But when McMahon, Duncan, Robbins, Clark, etc. attempt to explain Federal Theology, the FV people plainly point out that they have not been well-understood AT ALL. Wilson does NOT believe in justification by faith + works. He believes in justification by faith alone. Wilson DOES agree that Adam had to be PERFECTLY obedient in the Garden of Eden, in order to avoid the fall. So what's the huge stink? We are free to disagree with him regarding whether Adam could "merit" anything. But numerous (though certainly not all) Reformed theologians have questioned the place of "merit" in the Garden of Eden too, so even if Wilson is wrong, he is NOT outside the pale of orthodoxy. Wilson does NOT believe that ex opere operato salvific grace is conferred through the sacraments. If a person is baptized and/or partakes of the Lord's Supper, that does NOT automatically make that person right with Christ. Wilson is no Roman Catholic. Wilson uses phrases like "baptismal regeneration", that make us uncomfortable. And frankly, it may be unwise for him to throw such phrases around. Nevertheless, he means something very different by that phrase that Roman Catholics do. Let Wilson define himself, and his discomforting language suddenly proves itself to be nothing resembing a scandal. You may not like Wilson's paedocommunion. But I do! And so would Augustine, Cyprian, G.I. Williamson, Vern Poythress . . . At the end of the day, Wilson has said nothing that hasn't been said by other Reformed theologians. Wilson just says things in a way that gets people's feathers ruffled. Maybe Wilson goes too far with this. On the other hand, there are probably some people who need their feathers ruffled! But in any case, regardless of any agreements or disagreements, we should ALWAYS give a brother the benefit of the doubt, and do everything we can to TRY to understand him in a way that does NOT suggest "heresy". Far too many Reformed Theologians are "heresy hunters", just looking for someone to verbally burn at the stake. And THIS error is FAR WORSE that ANY doctrinal error Wilson may or may not have. Heresy hunting is a SIN. We should always assume the best, not the worst, and we should dialogue carefully with a brother, LONG before we finally toss in the towel and write off a brother as being an apostate heretic. I think Christ is ashamed of Robbins, McMahon, Duncan, Gerety, Clark, and anyone else who has jumped on the "FV is heresy" bandwagon. The body of Christ is hurt FAR worse by such slander, than it is hurt by any error the FV may have to offer. The distance between "FV" and "heresy" is so great that only a quick-tempered and uncharitable person would openly proclaim that the FV people are "going to hell", without having first had the courtesy to dialogue directly with them to make sure they have been clearly understood. McMahon said: Take note of the Scriptures, especially on serious issues - Proverbs 29:20 , "Do you see a man hasty in his words? There is more hope for a fool than for him." Such is the case when critiques are sent out that are unfair, or hasty in response. That is EXCELLENT advice! I wish Robbins, Gerety, McMahon, Clark, Duncan, etc. would FOLLOW that advice regarding the FV! I would be very interested to hear, for example, how much time ANY of these men spent dialoging directly with Doug Wilson? Early on, I spent a little time dialoging with Wilson's office, because there were some things I heard that truly concerned me. But my few questions were quickly cleared up quite to my satisfaction. --- And sadly, it is my genuine assumption that many of the men listed above didn't give Wilson the same courtesy. If I am wrong, then please correct me. If any of these men spent some serious time dialoging directly with Wilson before publicly blasting him, then I would be glad to hear that at least an attempt was made. Originally posted by StaunchPresbyterian Am I reading him wrong? (Disclaimer: I have read very little of Wilson's other works, and am merely discussing on the basis of his Credo quoted at the beginning of this thread . . which I supposed should be clear enough to make his point.) Joseph, do you think I have misread Wilson's Credo on Justification? Originally posted by StaunchPresbyterian Originally posted by StaunchPresbyterian Am I reading him wrong? (Disclaimer: I have read very little of Wilson's other works, and am merely discussing on the basis of his Credo quoted at the beginning of this thread . . which I supposed should be clear enough to make his point.) Joseph, do you think I have misread Wilson's Credo on Justification? -------------------- My response: ------------------- Originally posted by StaunchPresbyterian Well, I just finished reading this whole thread -- I find it interesting that Wilson's Credo on Justification cannot, on its own, settle the matter. Is not a creed written for the express purpose of being clear ? If so, Wilson has failed in this respect . . in my humble opinion. Personally, I think Wilson´s Credo on Justification is perfectly clear. I don´t have any difficulty understanding it (that I know of). However, I must respect the fact that Wilson´s Credo is not clear to you. No one can argue with that. Originally posted by StaunchPresbyterian And he certainly manifested a strange twist in it: I believe that God established two distinct covenants with mankind, one before the Fall, and one after. The first covenant was called a covenant of works in the Westminster Confession (7.2). I would prefer to call it a covenant of creational grace. The condition of covenant-keeping in this first covenant was to believe God´s grace, command, warnings, and promise. (Bold = mine) This is strange and novel (do I really have to say that?). The condition had little to do with believing and everything to do with obeying . Well, obeying involves believing, too . . this is true (as the first table of the Commandments makes clear). But, it wasn't merely that he believe, he had to obey! Wilson agrees 100% that Adam had to obey. A couple sentences after your quotation, Wilson does make it clear that "œPerfect and personal obedience" was required of Adam. Wilson´s only qualification is that "œeven for an unfallen man, is not possible unless he trusts in God´s goodness and grace." In other words, Wilson is not belaboring the point about the necessity of Adam´s perfect obedience in the original covenant, because it is a given . We all 100% agree on that. Wilson´s focus, therefore, is elsewhere. While he agrees that Adam had to perfectly obey, he wants to stress the point that perfect obedience was ONLY possible via a gift from God. And since this gift from God was undeserved, it can properly be called "œgrace". The word "œgrace" does not have to be used in the context of sin. It is a word that can be properly used anytime that a person is given something he has not earned. It simply means "œunmerited favor". I recently got an email from Mike Lawyer, an assistant of Doug Wilson. He was challenging my view of the Covenant of Works. He said: Just so you know, as I think you already do, Doug is all about grace. He´s all about receiving gracious things by faith. Not even Adam could have earned what cannot be earned. Salvation has always been a gift of God to undeserving people. For Adam it would have been a gift to a man who never sinned, for us it is a gift to people who are sinners by nature. I believe this quote from Doug Wilson´s assistant is the same thing we would hear from Doug Wilson´s mouth if he were to participate in this discussion. There is a similarity : Adam received grace. And we receive grace. There is also a difference : Adam received grace in a sinless state. But we receive grace in a sinful state. God has to condescend much further to offer us grace, than He did for Adam. Originally posted by StaunchPresbyterian Adam was a recipient of grace, and thus, the sin that plunged our race into death was a revolt against grace. Wilson seems to confuse creation and covenant here. On account of God having created Adam, he had to believe he existed, and he had to obey God with a personal, perpetual, and perfect obedience -- even without the covenant! Where do you see the word "œcovenant" in Wilson´s quote you give here? Even if you take the covenant totally out of the picture, Wilson´s argument still remains coherent. He is arguing thus: Adam did not deserve to be created in the first place. It was gracious for God to even give him his very existence. And if God gave him the natural ability to retain perfect holiness, this was a gracious gift as well, which Adam did not and could not have earned. Thus, covenant or no covenant, Adam´s very existence was necessarily in a state of grace "“ having received unmerited favor from his Creator. Thus, in this particular part of Wilson´s quote, I think any focus on the covenant itself is not necessary in order to understand Wilson´s thinking. Originally posted by StaunchPresbyterian The goodness of God (note: not the grace of God) is manifest in the "voluntary condescension." The point? Man always owed God obedience, but now God promised to bless that already-owed obedience! This is what expresses the goodness of God. Arguing from Wilson´s position, I think a person would have to say that your statement here just moves the question one step back. After all, did Adam earn or merit this reception of "œthe goodness of God"? Did Adam inherently deserve for God to create him and to be infinitely good to him? If not, then the word "œgrace" is still appropriate. Originally posted by StaunchPresbyterian So, Adam would have merited eternal life ex pacto , on account of the covenant. It wasn't that his works were inherently valuable, but that God was pleased, by "voluntary condescension" to attach a promised blessing to that obedience. When you say, "œIt wasn´t that his works were inherently valuable", you argue strongly for Wilson´s point. You admit that Adam could not truly merit anything before God. Then you go on to say that God voluntarily condescended to attach a promised blessing to the obedience. I think Wilson would heartily agree with you! But note your use of the phrase "˜voluntary condescension´. If it was "œvoluntary", then it must not have been required by strict (merit-based) justice. If it was a genuine "œcondescension", then it must not have been intrinsically required by strict (merit-based) justice. In other words, you are saying that Adam did not deserve for God to make this promise to him as a reward for obedience. Thus, you are saying that God was gracious to offer a reward in return for obedience. In short, you are saying that Adam could merit salvation from God, but only because God was gracious enough to put him in that position of ability. Now, that would be a funny way to state it. But it appears to me that this is just what you have said, Casey. So we need to get rid of that word "œmerit", or we need to get rid of the word "œgracious", or we need to get over it. Those are our three options. Originally posted by StaunchPresbyterian This is exactly what the Confession says (7.1): The distance between God and the creature is so great, that although reasonable creatures do owe obedience unto him as their Creator, yet they could never have any fruition of him as their blessedness and reward, but by some voluntary condescension on God's part, which he hath been pleased to express by way of covenant. I love that part of the WCF. Do you think Wilson disagrees with anything in that paragraph at all? If so, what? Originally posted by StaunchPresbyterian So, to call this a covenant of "creational grace" seems rather silly to me. His definition of this strikingly different covenant from the one the Confession describes. It denies that Adam could have obeyed on his own merits, How is his understanding different from the Confession? Even if Adam could have obeyed totally on his own, where did he get this ability? Did he get this ability on his own, or did God create him with this ability? Of course, we would say that God created him with this ability. Well, did Adam earn the right to be created with this ability? Did Adam merit his own creation? Clearly, it would be silly to suggest that a yet-uncreated-being can merit anything from God. God was gracious to give Adam existence in His image. Adam didn´t deserve to be created like that! Adam didn´t merit the right to be created any more than a baby merits the right to be conceived. Life is a gracious gift from God. You can´t earn physical life any more than you can earn spiritual life. Originally posted by StaunchPresbyterian and seems to confuse grace (prelapsarian/postlapsarian grace?). I am just unclear with what he means by "grace" here. How is God's grace to Adam any different from his grace towards us in Christ? Is Wilson differentiating the two? The second covenant is a covenant of redemptive grace. The thing that the two covenants have in common is grace, not works. No, he's not. That's what makes it confusing. Apparently grace is grace, and the same grace was in the first covenant that manifests itself in the second . . ? There is a similarity : Adam received grace. And we receive grace. There is also a difference : Adam received grace in a sinless state. But we receive grace in a sinful state. God has to condescend much further to offer us grace, than He did for Adam. God only had to condescend down to a perfect man then. But He now condescends all the way down to fallen men, who are infinitely lower than Adam initially was. Originally posted by StaunchPresbyterian "œPerfect and personal obedience," even for an unfallen man, is not possible unless he trusts in God´s goodness and grace. Wilson here seems rather clearly to deny the fourfold state of grace taught explicitly in the Confession's chapter on Free Will. I quote the Confession (9.2): Man, in his state of innocency, had freedom, and power to will and to do that which was good and well pleasing to God; but yet, mutably, so that he might fall from it. For Wilson, apparently Adam didn't have this power? Of course he had to obey God and believe in him, but is such the basis of his power to obey? No, the power to obey was not contingent on his obedience, but on the way God had created him. But did Adam merit being created that way? Did Adam deserve to be created with such an awesome ability? If not, then we still come back to the same fact of unmerited favor (grace) existing before the fall. Originally posted by StaunchPresbyterian The condition for keeping this covenant is the same as the first, although the circumstances are different. The condition always is to believe God. Wait a second. If, according to Wilson, grace was required in the Covenant of Works to enable Adam for obedience in order to obey that covenant . . And the conditions of both covenants are the same (obedience, I suppose?), then in the Covenant of Grace is God merely giving the same sort of grace he gave in the Covenant of Works to Adam? Is God merely enabling us to obey? This seems to be what Wilson is saying (but, it's very possible I'm misunderstanding him). I don´t think that is where Wilson is trying to go. First of all, there is a difference. While God gives grace both to Adam and to us, it is very significant to note that God´s condescension is MUCH farther now than it was then. So in one sense, grace is much bigger now, because it lifts us so much farther (because we start out so much lower than Adam.) The quality of the grace is the same, but the extent of the grace has to be much greater for us, in order to lift us out from the deep pit of sin we are in. Second of all, any Reformed theologian worth his salt DOES say that perfect sinless obedience is just as much a requirement for us as it was for Adam. But in our case, Christ keeps that requirement for us. Thank God that there is a Savior for us! But the presence of the Savior in no way removes that fact that perfect obedience is still required. The legal stipulations of the covenant aren´t any lighter for us than for Adam . . . it´s just that in our case there is someone else who meets the conditions for us, rather than we having to meet those conditions ourselves. These points are made, not to smuggle "œworks" from the covenant of works into the covenant of grace, but rather the opposite. I believe we must insist that autonomous works be banished from every human realm and endeavor, whether fallen or unfallen ( 1 Cor. 1:31 ). Seems to me he's trying to sit on that fence again. He's denying what he just tried to prove. Grace enables Adam for obedience. It's the same grace and same condition in the Covenant of Works as in the Covenant of Redemption. God enables us for obedience, by his grace, in the Covenant of Grace. Wilson is arguing that there is grace in both covenants. But he is not arguing that grace operates in precisely the same way in both covenants. I think Wilson is saying this: for Adam : God´s grace enabled Adam to obey. for you and me : God´s grace provided Christ to die in our place. God´s grace provided Christ to obey in our place. God´s grace caused us to trust in Christ. I do not think Wilson is making both covenants the same. He sees most of the same distinctions that you and I see between them. He is just arguing that the presence of grace is NOT one of the distinctions. Obedience was required of Adam. Trust in Christ´s obedience is required of us. But in both cases, only God´s grace could bring about the good result. Originally posted by StaunchPresbyterian Am I reading him wrong? (Disclaimer: I have read very little of Wilson's other works, and am merely discussing on the basis of his Credo quoted at the beginning of this thread . . which I supposed should be clear enough to make his point.) I don´t think you are anywhere near the error of those who anathematize Wilson. I think your understanding of Wilson´s Credo might have been a lot clearer if you had spent a lot of time reading other writings by Wilson. But since you have not done this, I think you raised some fair questions. I hope I understand Wilson correctly, and I hope I have helped clear up some of your questions about what Wilson is really saying. Please let me know whether I have succeeded or failed in this case. Casey, thank you for participating in this discussion, and THANK YOU for your EXCELLENT attitude!! Your brother in Christ, Joseph [Edited on 4-9-2006 by biblelighthouse] While we are on the topic of grace vs. merit, I would like to share some interesting quotes with you: John Calvin openly complained about the word "merit", saying, "œI wish that Christian writers had always exercised such restraint as not to take it into their heads needlessly to use such terms foreign to Scripture that would produce great offense and very little fruit" (Institutes 3.15.2.). Regarding Christ he writes, "œIt is absurd to set Christ´s merit against God´s mercy"¦ Apart from God´s good pleasure Christ could not merit anything " (Institutes 2.17.1.). Of Adam´s position in the garden of Eden, Turretin wrote, "œ Adam"¦would not have merited life in strict justice , although (through a certain condescension) God promised him by a covenant life under the condition of perfect obedience" (Institutes 17.5.7.). Anthony Burgess (A Westminster delegate) could say of the pre-fall situation, "œthough it were a Covenant of Works, it cannot be said to be a covenant of merit " (Quoted in Garver's essay, "œThe Covenant of Works in the Reformed Tradition," available at http://www.joelgarver.com ). Theologian without number could be multiplied that made these same reservations. . . . Rowland Ward , who has probably amassed a greater amount of resources than any other recent historian regarding the Reformed doctrine of the covenant of works, has argued that its administration was almost always seen as gracious, and its rewards as unmerited (See his recent God and Adam ). If Adam would not have been justified by his obedience, then we have to assume that Christ did not obtain justification for us with His obedience. I am not sure that is valid. Type vs. Antitype works within a biblical framework as far as Federal headship goes, but Adam was able to fall, Christ, was not. We are justified by Christ's works, and He Himself is justified by his own works. The very nature of His human works are rooted in that He was without sin and had a divine nature as well. Joh 5:36 But the testimony that I have is greater than that of John. For the works that the Father has given me to accomplish, the very works that I am doing, bear witness about me that the Father has sent me. Joh 10:32 Jesus answered them, "I have shown you many good works from the Father; for which of them are you going to stone me?" Joh 10:37 If I am not doing the works of my Father, then do not believe me; Joh 10:38 but if I do them, even though you do not believe me, believe the works, that you may know and understand that the Father is in me and I am in the Father." Joh 14:10 Do you not believe that I am in the Father and the Father is in me? The words that I say to you I do not speak on my own authority, but the Father who dwells in me does his works. And we perform good works through the power of the indwelling Spirit, not our flesh: Joh 14:12 "Truly, truly, I say to you, whoever believes in me will also do the works that I do; and greater works than these will he do, because I am going to the Father. I have always thought Christ was the only man who could be justifies by his works . . is that wrong ? [Edited on 3-16-2006 by Saiph] Mark, I would say the credo is a good example of confusion leading to problems. It most often comes from a sdesire to restate "better" classical formulations. What do I mean? The initial two paragraphs are very good, and I say, "Amen!" I believe that Jesus Christ was justified by God in His resurrection from the dead, being declared with power to be the Son of God ( Rom. 1:4 ). He was justified in the Spirit ( 1 Tim. 3:16 ), vindicated by God, and exalted to the right hand of God the Father. This justification, along with Christ´s active and passive obedience , and all His other perfections, is imputed to His people , and is the only basis for all that they have in Him. This justification of Christ, this resurrection from the dead, was for our justification ( Rom. 4:25 ). I believe that God in His sovereign and secret decree has elected by name a countless number to eternal salvation ( Eph. 1:11 ). Each of these elect are justified individually, and irreversibly, at the point of their conversion, when God imputes to them all the righteousness of Jesus Christ ( Rom. 8:29-30 ). The ground of this justification is the righteousness of Jesus Christ, plus nothing , and is appropriated by the instrument of faith alone, plus nothing, and even this faith is to be understood as a gift of God, so that no one can boast ( Eph. 2:8-10 ). The problem comes in that as soon as Wilson speaks of a "Covenant of Creational Grace," he is (unwittingly?) militating against those paragraphs. How? If the first Covenant was one of grace, not works, then how does Christ come by obedience that is imputed to us? what is the active obedience that Wilson speaks of that is imputed? It can't be fulfillment of the Covenant, since Wilson also says that Adam would not have earned anything by his obedience. Classical Reformed theology says that God graciously determined to make a covenant (where He did not have to) and to grant a reward upon obedience (which He again did not have to do), but once He did make that Covenant, He was bound by His oath. Further, I think that this statement is at least unclear: "Perfect and personal obedience," even for an unfallen man, is not possible unless he trusts in God´s goodness and grace I don't see the Scriptures ever calling unfallen man, or glorified man to trust in God's grace (which presumes demerit), but rather to trust in God Himself. A small difference perhaps, but significant. This credo expresses well for me where Wilson is - I think he is orthodox in his main doctrine, and especially on imputation and justification. But he too often (and in too many areas) desires to be "cute" or novel and that makes things less clear instead of more. Not a good thing. I don't think a blanket condemnation of Wilson is in order. Instead we should affirm what is good in what he says, and at the same time warn that he is a confusing teacher at times. I hope I am coming accross as balanced and not strident, because that is how I feel at the moment. If Wilson is being 'cute', and knows the difference between orthodoxy and illicit in regards to justification, shame on him for being cryptic to the confusion of Gods saints! Personally, I know that Wilson knows the difference; sitting on the fence for the sake of being contemprary is just plain irresponsible, not at all cute. The overseeing body here on this forum is the RPCGA; Federal Vision is heresy. As has been said before, Wilson has elbowed up to the discipline; if he does not embrace it, it would behoove him to come out and say so, so as not to endanger Gods people whom look to his wisdom; I won't hold my breath......... Joseph, The board has no official rule per se on discussing Wilson. However, we are against components of the discussions that promote the heresy. Draw the line as you see fit, but be cautios as we will be watching. [Edited on 4-9-2006 by Scott Bushey] Originally posted by Scott Bushey If Wilson is being 'cute', and knows the difference between orthodoxy and illicit in regards to justification, shame on him for being cryptic to the confusion of Gods saints! Wilson is not cryptic. He has said nothing unorthodox concerning justification. He believes in sola fide like you and me. There just happen to be a number of people (such as yourself) who have a "Ready, Fire, Aim" mentality. Instead of being responsible, you just lambast your opponent before you even bother to understand what he's saying.	https://www.puritanboard.com/threads/a-short-credo-on-justification-douglas-wilson.12528/page-3#post-170882
Materials \| Free Full-Text \| Long-Term Atmospheric Corrosion Behavior of Epoxy Prime Coated Aluminum Alloy 7075-T6 in Coastal Environment The atmospheric corrosion of epoxy prime coated aluminum alloy 7075-T6 exposed for 7, 12 and 20 years was investigated. The remaining thicknesses of epoxy prime coatings for macroscopically intact coating areas followed a normal distribution and decreased linearly. EIS results demonstrated that the corrosion resistance of the coating decreased with exposure time. After 20 years of exposure, the epoxy coating had lost its protection as cracks existed within the coating and exfoliation corrosion had occurred on the substrate. The substrate was sensitive to exfoliation corrosion through metallographic and TEM analysis. The corrosion products were mainly hydroxides of aluminum. The morphology and chemical compositions of the coating bubbling area and propagation characterizations of exfoliation corrosion were analyzed by SEM, EPMA and EDS. Cracks between the lumps of corrosion products provided the channels for the transmission of corrosion mediums. Furthermore, the mechanical model was proposed to analyze the propagation characterization of exfoliation corrosion. Long-Term Atmospheric Corrosion Behavior of Epoxy Prime Coated Aluminum Alloy 7075-T6 in Coastal Environment by Sheng Zhang 1 , Yuting He 1,* , Teng Zhang 1 , Guirong Wang 2 and Xu Du 1 1 Aeronautics Engineering College, Air Force Engineering University, Xi’an 710038, China 2 College of Materials Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China * Author to whom correspondence should be addressed. Materials 2018 , 11 (6), 965; https://doi.org/10.3390/ma11060965 Received: 5 April 2018 / Revised: 17 May 2018 / Accepted: 5 June 2018 / Published: 7 June 2018 Abstract epoxy prime ; aluminum ; EIS ; EPMA ; atmospheric corrosion ; exfoliation corrosion 1. Introduction Because of its high strength and low density, the aluminum alloy 7075-T6 is widely used in aircraft structures; however, the service of aircraft structures in the outdoor atmosphere exposes them to the influence of atmospheric corrosion [ 1 ]. To improve the corrosion resistance and safety of aircraft structures, coating systems have been extensively used to protect aluminum alloys against corrosion [ 2 , 3 ]. Generally, aircrafts utilize a three-layer coating system including a conversion coating, primer and topcoat [ 4 ]. Currently, the internal structures of military aircrafts mostly utilize the two-layer coating system composed of an anodic oxide film and an epoxy primer. The advantage of epoxy primer is that when water seeps into the coating, the active pigments rapidly hydrolyze, forming ions that passivate the metal surface, effectively preventing metal corrosion [ 5 , 6 ]. On the other hand, although these coating systems initially blocked the diffusion of ions effectively, with the increase of service time, water and aggressive ions gradually penetrated to the metal/coating interface, resulting in substrate corrosion and coating failure [ 7 ]. The corrosion of coated metals, organic coatings, and aluminum alloys has been extensively investigated through accelerated tests or electrochemical methods [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. These laboratory tests are particularly useful for determining the influence of specific pollutants and ions [ 15 ], but for evaluating the service performance of structures or materials, field testing in the actual environment is more reliable. Smith et al. reported that the corrosion of a bare 7075-T6 aluminum alloy sheet exposed for 4 years was mild in marine and inland atmospheres [ 16 ]. Sun et al. carried out atmospheric corrosion tests on the alclad 7075 aluminum alloy in four different environments in China for 20 years and investigated the mechanical properties and depth of pitting [ 17 ]. Katayama et al. investigated the corrosion properties of the carbon steel plates with Zn, Al and Zn-Al thermally sprayed coatings exposed to a coastal area for about 33 years using electrochemical impedance measurements [ 18 ]. Different kinds of epoxy coating systems were exposed to the corrosive atmosphere of petrochemical industry for 2 years to evaluate their protective effects and determine the corrosive grade of the corrosive atmosphere [ 19 ]. Bano et al. reported the performance of epoxy-polyamide primer and coal tar epoxy topcoat system applied on mild steel which subjected to various natural exposures including marine, industrial and urban test sites of Karachi city for 21 months, while an accelerated test was also conducted for comparison [ 20 ]. In order to investigate the corrosion failure characteristic of airplanes served in the tropical coastal environment of Fiji for 1600 flight hours, epoxy prime coated aluminum alloy taken from the airplane was studied by examining the macro and micro images, and analyzing the composition of corrosion products [ 21 ]. The results of these atmospheric exposure tests are exceedingly valuable, and have provided the academic and industrial communities with important data; however, there are very few reports about the corrosion behavior of epoxy primer coated aluminum alloys exposed to a coastal environment for a long period of time. This paper presents the surface appearance and corrosion products of epoxy primer coated aluminum alloy 7075-T6 exposed for 7, 12 and 20 years in the Wanning test site in China. We also estimated the atmospheric corrosion properties of epoxy coatings using a statistical method and electrochemical impedance measurements. The exfoliation corrosion resistance of the aluminum alloy substrate was analyzed by metallographic and TEM analysis. Finally, we analyzed the morphology and chemical compositions of coating bubbling area and propagation characterizations of exfoliation corrosion by SEM, EPMA and EDS. 2. Experimental 2.1. Specimens Figure 1 shows the geometry of the specimens exposed to the coastal environment. The intermediate section of the specimen was the corrosion test section with a 220 mm length, 400 mm width, and 4 mm thickness. Extruded 7075-T6 aluminum alloy was selected as the substrate and its chemical composition is given in Table 1 . The three dimensions of a microstructure are generally defined as longitudinal (L), long transverse (T), and short transverse (S) and the L direction is identified as the direction of extrusion. The substrates were ultrasonically degreased in acetone, anodized in sulfuric acid, and sealed with hot water. The ultimate thickness of the anodic oxide film was 8 μm. Finally, the zinc chromate pigmented epoxy primer was sprayed within 24 h and the surface of the specimen to be sprayed was perpendicular to the spray gun during spraying. Then the coated specimens were left to dry in a ventilated environment at 40 °C. The average thickness of the coating was controlled at 50 μm. The zinc chromate is a kind of typical anti-corrosion pigments in the coating. Although the epoxy zinc chromate primer had been banned, the epoxy zinc chromate primer was widely applied to protecting the internal aluminum alloy structures of military aircrafts against corrosion before 1997. The research of this primer is of great significance for evaluating the structural safety of aging aircraft serving in coastal environments for a long period of time. Figure 1. Schematic drawing of the specimen’s test section. Table 1. Chemical composition (wt %) of extruded 7075-T6. 2.2. Field Exposure Test The exposure tests were performed in the Wanning test site in China. Figure 2 shows the location of the Wanning test site. Table 2 provides the average environmental data measured in 1997 at Wanning and the annual environmental data can be found free in this website named “China Gateway to Corrosion and Protection” whose URL is http://data.ecorr.org/edata/01/0103/010301/01030101/index.html . All the specimens were placed on the test racks facing south at an angle of 45° in a ventilated room to simulate the environment in which the internal structures of an aircraft were located. The analyses were then carried out on the upper surface, facing the sky, since it had endured more pronounced aging and corrosion. Five specimens were withdrawn from the test sites after 7, 12 and 20 years of exposure for each time period and then kept in a desiccator. Figure 2. Location of the exposure test site. Table 2. Average environmental data measured in 1997 at Wanning. 2.3. Surface and Corrosion Products Analyses The upper surface appearance of the test section (defined in Figure 1 ) of the specimens with different years exposure was analyzed using a PXS-5T stereomicroscope with an image sensor. After microscopic observation, the upper surface corrosion products in substrate corrosion area of a specimen exposed for 20 years were identified by X-ray diffraction using an X’Pert PRO. 2.4. Electrochemical Impedance Measurement Electrochemical impedance spectroscopy (EIS) measurements were performed using a PARSTAT 2273 electrochemical workstation (AMETEK, Princeton, NJ, USA). The electrochemical impedance test samples with a coating area of 1 cm 2 were taken from the macroscopically intact coating area on the test section of the specimens. A three-electrode cell arrangement was used in the experiments, with the coated sample electrode as working electrode (WE), a platinum wire as counter electrode (CE) and a saturated calomel electrode (SCE) as reference electrode (RE). The electrolyte employed in this measurement was a 3.5% NaCl solution. The impedance data was taken after measuring the open circuit potential for several tens of seconds. The impedances were characterized over frequencies ranging from 100 kHz to 10 mHz, using a 20 mV perturbation signal. Equivalent circuit modeling was performed using ZSimpWin software (EChem Software, Ann Arbor, MI, USA). 2.5. Sectional Analyses 2.5.1. Measurement of Thickness of Coating The remaining thickness of the epoxy coating in the macroscopically intact coating area on the test section of the specimens after different exposure times was estimated using a statistical method. Slices of coating cross-section from a macroscopically intact area within the test section were ground with a 1000 grit SiC paper. The remaining thickness of the upper surface coating was measured by RH-8800 digital video microscope (HIROX, Tokyo, Japan) and TSview-7 software. Figure 3 shows the cross-sections of one side of the upper surface coating of specimens exposed for different years. For every corrosion period, 6 sections were measured, each of which was divided in 10 evenly spaced regions to measure the remaining thickness of the coating. Figure 3. Cross-sections of specimens exposed for different years (one side of the upper surface coatings): ( a ) 7 years; ( b ) 12 years; ( c ) 20 years. 2.5.2. Analysis of Exfoliation Corrosion Resistance of Aluminum Alloy Substrate The S-L section of the extruded 7075 aluminum alloy substrate exposed for 20 years was ground with a 1000 grit SiC paper, polished with 2.5 μm diamond paste, and chemically etched with Keller’s reagent (1.0 mL HF + 2.5 mL HNO 3 + 1.5 mL HCl + 95 mL H 2 O) for 15 s. The microstructure of the S-L section was then observed by metalloscopy. A coating test sample with a cross-section thickness of 0.5 mm from a specimen that had been exposed for 20 years was ground with a 2000 grit SiC paper until a thickness of 80 μm was reached (measured with micrometer caliper) was obtained. Thinning and perforation were carried out on 695 PIPS COOL ion beam thinner (Gatan, Pleasanton, CA, USA), thus completing the preparation of TEM samples. The grain boundary structure of the aluminum alloy was observed on a FEI Talos F200X high resolution transmission electron microscope (FEI, Hillsboro, OR, USA). 2.5.3. Corrosion Failure Analysis of Aluminum Alloy Coating System The S-L sections of the coating bubbling area and of the junction area between the corroded and un-corroded substrate of a specimen exposed for 20 years were ground with a 1000 grit SiC paper and polished with 2.5 μm diamond paste. The sectional morphology and chemical compositions of the corrosion products were analyzed through SEM and EDS. In addition, the elemental distribution of the coating bubbling section was obtained by EPMA. 3. Results and Discussion 3.1. Surface Appearance and Characterization The surface appearance of coated specimens exposed for 7, 12 and 20 years in the Wanning test site are shown in Figure 4 . The degree of aging increased with the exposure time and the color of the coating changed, gradually becoming darker. After 7 years a small amount of salts and impurities were deposited on the surface of the coating ( Figure 4 a); the deposit present on samples that had been exposed for 12 and 20 years also contained impurities such as sand and dust ( Figure 4 b,c). The concentration of impurities was higher on samples exposed to the atmosphere for 20 years. The surface of the epoxy coating remained macroscopically intact when the exposure times was lower than or equal to 12 year, and no localized corrosion occurred. On the other hand, after 20 years the epoxy coating had partially lost its protection ability, and both the coating and the anodic oxide film had been completely destroyed in the exfoliation corrosion regions ( Figure 4 d). Meanwhile, the extruded 7075 aluminum alloy substrate displayed layered peeling and the formation of white corrosion products in box A, which are typical of exfoliation corrosion ( Figure 4 c). The percentage of corrosion regions in corrosion test section of the most severely corroded specimen is up to 70% after 20 years exposure. At the same time, the average percentage of five specimens is about 30%, which is similar to that of specimen 2# ( Figure 4 d). Figure 4. Surface appearance of coated test specimens exposed to the Wanning test site: ( a ) 7 years; ( b ) 12 years; ( c ) 20 years; ( d ) 20 years (whole upper surface). The XRD pattern of the white corrosion products in substrate corrosion area of a specimen exposed for 20 years is shown in Figure 5 . The results show that the corrosion products were comprised of Al(OH) 3 . The volume of this component is larger than that of the parent material, thus forcing the sheets apart. In addition, the more elongated the grains are, the greater the generated wedging force [ 1 , 22 , 23 , 24 ]. Figure 5. XRD spectrum of exfoliation corrosion products. 3.2. Statistical Analysis of the Remaining Thickness of Coating From Figure 3 , it can be observed that the thicknesses of the epoxy coatings decreased noticeably with exposure time, and the outer surface of the coating was smooth, without visible damage. The remaining thicknesses were described through the Normal, Log-normal, Gumbel, Logistic and Weibull distributions. The corresponding distribution functions are shown below: (1) Normal distribution: F ( x ) = ∫ − σ − ( x ) = ∫ − ∞ lg x 1 2 π ⋅ σ ⋅ e − ( lg x − 2 2 σ 2 d x (2) (3) Gumbel distribution: F ( x ) = exp [ − exp ( − x σ ) ] (4) Logistic distribution: F ( x ) = exp [ ( x − μ ) / σ ] exp [ ( x − μ ) / σ ] (4) (5) Weibull distribution: F ( x ) = 1 − exp [ − ( x σ ) β ] (5) where x is the remaining thickness variable; μ is the location parameter which determines the range of abscissa value of the distribution function and indicates the location of the centralized distribution of measured data; σ is the scale parameter which determines the scale of the distribution function and indicates the discreteness of measured data; β is the shape parameter which determines the shape of the distribution function and directly changes the property of the distribution function. Changes of σ only compress or expand the distribution function without changing its basic shape. After the logarithmic linearization of these five distributions, the different parameter values were obtained by the least square fitting [ 25 ] and the fitting degrees were compared through the Pearson correlation coefficient ( r ). Table 3 shows the fitting parameters and correlation coefficients corresponding to the different distributions. Table 3. Distribution parameters and correlation coefficients of the distribution models. According to the relationship between the correlation coefficient and the t -distribution, the critical correlation coefficient ( r c ) that satisfies distribution assumption can be calculated by [ 26 ]: r c = t α ( n − 2 ) ( n − 2 ) + t α 2 ( n − 2 ) (6) When the sample size ( n ) is 60 and the significance level α is 0.01, the r c is 0.330. It can be seen in Table 3 that all the distributions have a good correlation with the remaining thickness; however, the data is fitted most effectively by the normal distribution function, while the Gumbel distribution describes the data least effectively. The probability paper test of normal distribution of the remaining thickness is shown in Figure 6 and these data for 20 years are from areas where coating still existed (about 70% of the surface). According to the results, it can be concluded that the remaining thickness of the epoxy coatings exposed over 7 years satisfies a normal distribution. Figure 6. Result of the probability paper test of normal distribution (20 years’ data from coating-still-existing areas). The remaining thickness to be considered for the practical application of the coating was then determined by defining a reliability ( p s ) and confidence level ( γ ). For fixed values of p s and γ , the remaining thickness ( t ) can be calculated by: t = t ¯ + k 1 n ∑ t i + k [ ∑ t i 2 − n ( 1 n ∑ t i ) − 1 ] 0.5 (7) In Equation (7), t ¯ is the thickness average, s is the thickness standard deviation, n is the sample size, and k is a one-side tolerance coefficient that can be calculated using the methods reported previously [ 26 ]. In this paper, n is 60, and k is −3.237 corresponding to the general reliability of 99.9% and confidence level of 95%. The estimated thickness of the epoxy coating exposed for 7 years is 39.5 μm, as calculated by Equation (7). This means there is a 95% probability that the coating thickness will be over 39.5 μm in more than 99.9% of the regions after 7 years of exposure [ 27 ]. Figure 7 shows the average remaining thickness and the remaining thickness with 99.9% reliability and 95% confidence level of epoxy coatings in the macroscopically intact coating area on the test section of the specimens exposed for different times. The average data in Figure 7 means the arithmetic mean value of the measured thickness data and these data for 20 years are from areas where coating still existed. Figure 7. Relationship between atmospheric corrosion exposure years and remaining thickness (20 years’ data from coating-still-existing areas). It can be seen that the remaining thicknesses of epoxy coatings decrease linearly as the exposure time increases ( Figure 7 ). As those thickness data for 20 years are from areas where coating still existed, this linear degradation relationship is only valid for macroscopically intact coating areas. This linear degradation phenomenon may be mainly due to the fact that yearly variations of the atmospheric environment are negligible, although there are seasonal and daily variations. Thus the rate of aging and corrosion and of thickness decrease of the coating was constant. 3.3. Electrochemical Characterization of Coated Samples with Different Exposure Years The experimental Bode plots of coated samples after different exposure periods are shown in Figure 8 , where the experimental data (scatter plot) can be seen to be accurately described by the fitted data (curves). The Bode log \|Z\| and the Bode phase angle plots can better explain the frequency specific impedance behavior. From the Bode log \|Z\| plots ( Figure 8 a), it may be observed that the oblique lines of the impedance curves at high frequency, corresponding to the coating capacitance, shift towards lower frequencies with the exposure time. This result indicates that the capacitance of the coating increased and its corrosion resistance decreased with the increase in exposure time. In addition, as the coastal exposure time increased, the values of the impedance at 10 mHz, \|Z\| f=0.01 , decreased significantly from 70.8 MΩ cm 2 to 0.075 MΩ cm 2 . After 12 years of exposure, the impedance of the coating still had a higher value, and the epoxy zinc chromate coating had a protective effect on substrate. On the contrary, after 20 years of exposure, the phase angle was less than zero at the low frequency region ( Figure 8 b). This phenomenon shows the inductive characteristics, and indicates that the coating has lost its capability of protecting the substrate. Figure 8. EIS results and its fitted data of coated specimens exposed for 7, 12, and 20 years: ( a ) Bode log \|Z\| versus log (f/Hz) plots; ( b ) Bode phase angle versus log (f/Hz) plots. The Bode log \|Z\| plots in Figure 8 a are all described by lines with slopes between −0.5 and −0.2 in the middle frequency section, where a linear platform should appear. This characteristic of the coated specimen usually occurs in the middle stage of immersion and is called the Warburg impedance characteristic [ 28 ]. The production of Warburg impedance is due to the resistance of additive particles in organic coatings: the electrolyte seeps into the interior from gaps between particles along the winding routes, rather than along the direction of the concentration gradient. This phenomenon is also known as ‘tangent direction diffusion’ [ 29 ]. According to the characteristics of the epoxy zinc chromate coating, the phenomenon may be due to the addition of zinc chrome pigments into the coating. When the electrolyte seeps into the coating, zinc chromates will hydrolyze and generate K 2 CrO 4 , ZnCrO 4 , Zn(OH) 2 and Zn 2 (OH) 2 CrO 4 . A large number of zinc chromate pigments and their hydrolysis products contribute to increase the transport resistance, making it more difficult for the electrolyte to seep into the coating and slowing down the transmission of reaction particles to the corrosion interface, thus causing the diffusion impedance [ 13 ]. The Bode phase angle plots of coated samples exposed for 12 and 20 years showed the existence of three time constants. When the electrolyte reached the anodic oxide film surface through the coating, the chlorine ions accelerated the dissolution of the anodic oxide film, and the corrosion of the aluminum alloy substrate occurred at the site of the destruction of anodic oxide film [ 30 ]. Therefore, the three time constants corresponded to the time constant of the impedance of coating, the time constant of the dissolution of the anodic oxide film, and the time constant of the corrosion of the aluminum alloy substrate. Although the compositions of the coated samples after exposure for 7, 12, 20 of years were basically the same, since the EIS results of samples exposed for 20 years showed inductance characteristics it became necessary to add inductance and inductive resistance in the equivalent electrical circuit selection of samples exposed for 20 years [ 31 ]. The equivalent circuits that were found to most closely reproduce the impedance diagrams are shown in Figure 9 . The curves were fitted using the ZSimpWin software. The elements of the two chosen equivalent electrical circuits are as follows: R s is the solution resistance, R c and Q c are the coating resistance and coating capacitance, respectively, and W represents the Warburg diffusion impedance of the coating. The parameter groups Q f and R f represent the interface double layer capacitance and the charge transfer resistance of anodic oxide film corrosion reaction, respectively. The parameter groups C dl and R t represent the charge-transfer reaction at the electrolyte/substrate aluminum alloy. L and R L respectively represent the inductance and inductive resistance produced by substrate corrosion. The inductive loop is usually obtained by the corrosion products deposited or adsorbed on the sample surface [ 32 , 33 ]. Figure 9. Electrochemical equivalent circuits for fitting the impedance data: ( a ) 7 and 12 years; ( b ) 20 years. In this work, the frequency response characteristics of the coating and anodic oxide film were not consistent with the pure capacitance; therefore, the capacitance elements were fitted by constant phase elements Q (CPE) to compensate for non-ideal capacitance [ 29 ]. The impedance of Q is given by the following equation [ 18 , 34 ]. Z Q = Y 0 − 1 ( j ω n ( ≤ n ≤ ) (8) In Equation (8), Y 0 is the CPE-constant, j is the imaginary number unit, ω is the angular frequency ( ω = 2 πf , f being the frequency), and n is the CPE-power. When n = 1, the CPE represents a pure capacitive. Table 4 shows the EIS results analyzed by ZSimpWin. It can be observed that R c , R f and R t decreased with the exposure time. With the aging and corrosion of the coating, chlorine ions and other aggressive ions were more prone to invade the anodic oxide film. The long-term action of aggressive ions led to the local dissolution and destruction of the anodic oxide film, which made the corrosion of the substrate easier, and thus the R t decreased. The series inductive resistance, R l , of the coated samples exposed for 20 years was 6.99 × 10 4 Ω cm 2 , which indicated that the corrosion products deposited on substrate surface had a certain protective effect on the aluminum alloy substrate. Table 4. Simulated parameters of EIS results of coated samples exposed for 7, 12, and 20 years. Figure 10 shows the backscattered electron image of the S-L section of the specimen that was exposed for 20 years. Although there was no visible damage along part of the sectional coating thickness, obvious signs of cracking appeared between the local coating and the anodic oxide film and within the coating, leading to a reduced resistance of the coating to the infiltration of water, chlorine ions, sulfate ions and other aggressive mediums, and thus leaving the substrate very vulnerable to corrosion. This was a good explanation for the apparently contrasting observations made on the coating exposed for 20 years of an apparently intact structure but inductive EIS characteristics. The main causes of cracking were: Figure 10. Backscattered electron image of the S-L section of the specimen exposed for 20 years: ( a ) SEM morphology; ( b ) local enlarged SEM morphology. (1) Cathodic disbondment: Under the conditions of atmospheric corrosion, the anodic oxide film or aluminum alloy substrate under the coating endures electrochemical corrosion. The oxygen reduction reaction (Equation (9)) occurs on the cathode and the pH in the cathode reign increases significantly. The strong basic environment damages the metal oxide or polymer-coating of the interface, which affects the bonding of the coating to the anodic oxide film, and then causes the cracking [ 35 , 36 , 37 ]. (9) (2) Photoaging of coatings: Small molecules such as ketones, alcohols and acids can be washed away by water during photoaging (ultraviolet irradiation), continuously changing the composition of the coating, which therefore contracts and is subject to a decrease in thickness. This causes and embrittlement of the coating [ 38 ]. In addition, the loss of the polymer from the coating will effectively increase the volume concentration of the pigments on the surface of coating. Thus, the surface of coating will become relatively brittle while the inner layer of coating is still relatively elastic, which leads to the superficial and inner cracking of the coating. (3) Temperature alterations: The seasonal and daily alternations of atmospheric temperature cause the expansion and contraction of the coating, and the change of coating tension and internal stress, which leads to the decrease of adhesion, cracking and destruction of coating [ 39 ]. (4) Effect of SO 2 air pollutant: The air pollutant SO 2 can significantly reduce the adhesion of the coating, resulting in cracking between the coating and anodic oxide film. When SO 2 and H 2 O are simultaneously present, the destruction of the coating is more obvious [ 40 ]. 3.4. Analysis of the S-L Section of Coating Bubbling Area The S-L sectional SEM morphology and EDS spectrum of the corrosion products and the elemental distribution in the coating bubbling area of coated specimen exposed for 20 years are shown in Figure 11 . It can be seen that there was an evident bubbling area in the S-L section of the coated specimen exposed for 20 years ( Figure 11 a). The anodic oxide film under the bubbling area had been destroyed and the aluminum alloy substrate had also been corroded. The EDS results ( Figure 11 b) and elemental distributions ( Figure 11 c,d) revealed that the corrosion products in the interior of the bubbling area of Figure 11 a were mainly hydroxides of aluminum. The accumulation of corrosion products between the coating and the substrate and the expansion of their own volume led to the bubbling of the local coating. Sulphur was present throughout the entire coating area, as shown in Figure 11 e. The only source of S was the air pollutant SO 2 . The presence of SO 2 significantly reduced the bonding force between the coating and the substrate, and also reduced the corrosion resistance of the coating [ 40 ]. The presence of chlorine in the corrosion products was seen from Figure 11 b. The existence of the chlorine ion accelerated the dissolution of the anodic oxide film [ 41 ]. Figure 11. S-L sectional analysis in the coating bubbling area of coated specimen exposed for 20 years: ( a ) SEM morphology; ( b ) EDS spectrum of the corrosion products; ( c – f ) the elemental distributions of O, Al, S and C obtained by EPMA. In Figure 11 f, the epoxy coating, represented by the presence of carbon, was divided into two parts: an upper part and a lower part. This phenomenon may be due to a certain degree of cracking partaking place between the two layers of the inner coating before the corrosion of the aluminum alloy substrate, as shown in Figure 10 . After the corrosion of the substrate occurred, the corrosion products moved along the cracks and inside the coating. The coating was then divided into two parts due to the accumulation and expansion of the corrosion products. 3.5. Analysis of Exfoliation Corrosion of the Aluminum Alloy Substrate 3.5.1. Exfoliation Corrosion Resistance Analysis of the Aluminum Alloy Substrate The metallurgical structure of the S-L section and the grain boundary microstructure of 7075-T6 aluminum alloy substrate exposed for 20 years are shown in Figure 12 . The grains of extruded 7075-T6 aluminum alloy were flat in the direction of extrusion, and there were a large number of second phase particles ( Figure 12 a). The second phase particles of 7075 aluminum alloy mainly include η(MgZn 2 ), T(AlZnMgCu), S(CuMgAl 2 ) and β(Al 7 Cu 2 Fe). The main strengthening phases of 7××× aluminum alloy after T6 aging are the G. P. zone and a small amount of transitional phase η ′ (MgZn 2 ). A continuous chain of precipitates is distributed at the grain boundary ( Figure 12 b). From the comparison of EDS analysis results of the intragranular α solid solution (Area 2#) and of the precipitates at the grain boundaries (Area 1#), a higher Mg and Zn content is noticed in the grain boundary precipitates, and thus it can be inferred that the grain boundary precipitates consisted mainly of the η phase. Figure 12. 7075-T6 aluminum alloy ( a ) S-L metallographic section; ( b ) grain boundary structure and EDS analysis taken by TEM. The η phase in the pitting process acts as an anode and will be dissolved preferentially [ 42 ]. As the continuous chain of the η phase is distributed at the grain boundary, the anodic η phase at the grain boundary and the precipitate free zone (PFZ) form corrosion interactions and corrosion active channels, resulting in a high susceptibility to intergranular corrosion [ 43 ]. Under the action of corrosive mediums, the corrosion propagates along the grain boundaries parallel to the direction of extrusion, and corrosion products accumulate at the grain boundaries. Due to the expansion of corrosion products, wedge forces are generated at the grain boundaries, and eventually the surface of the alloy is exfoliated. Therefore, the 7075-T6 aluminum alloy substrate with 20 years exposure is very sensitive to exfoliation corrosion. 3.5.2. Propagation Characterization Analysis of Exfoliation Corrosion The junction area between the corroded and un-corroded substrate can be used to study the propagation of exfoliation corrosion. Figure 13 shows the S-L sectional SEM morphology and EDS spectrum of corrosion products of the coated specimen that was exposed for 20 years. The substrate and epoxy coating in the left of the exfoliation corrosion area were intact and did not present coating bubbling ( Figure 13 a). The exfoliation corrosion of the S-L section propagated along the direction of extrusion (L direction). Due to the expansion of intergranular corrosion products, the corrosion products of the A area in Figure 13 b showed a wedge shape. Moreover, the structures of the aluminum alloy on the upper side of the corrosion product area noticeably exfoliated outwardly, whereas the exfoliation of structures on the lower side was not obvious. At the same time, there was an obvious crack at the tip of the corrosion products. Figure 13. S-L sectional analysis in the junction area between the corroded and un-corroded substrate of the specimen exposed for 20 years: ( a ) SEM morphology; ( b ) local enlarged SEM morphology; ( c ) EDS spectrum in the lump area of the corrosion products; ( d ) EDS spectrum in the cracking area of the corrosion products. Due to the ventilation and daily temperature alternations on the test site, the surface of the specimens underwent dry and wet alternation, which made the products of intergranular corrosion harden ( Figure 13 c). From the results of EDS analysis in the block area of corrosion product in Figure 13 c, it can be found that C and S appeared in the lump area of corrosion products besides the oxygen and the elements contained in the aluminum alloy substrate. Carbon may originate from the organic coating. After local aging and destruction of the organic coating, the polymers entered the corrosion area with the corrosive medium. The existence of S indicated that the air pollutant SO 2 was indirectly involved in intergranular corrosion through conversion to acid in the moist environment. The annual average of NO 2 in the Wanning test site was far less than the annual average of SO 2 , as shown in Table 2 , correspondingly, N was not found in the corrosion products ( Figure 13 c,d). Thus, it can be preliminarily inferred that NO x was not involved in the exfoliation corrosion of the aluminum alloy substrate. The EDS analysis results of the cracking area ( Figure 13 d) showed the presence of Na and Cl, which were not measured in the lump area ( Figure 13 c). This result indicated that the cracks in the area of the corrosion products provided the channels for the transmission of corrosion mediums such as NaCl, which created conditions for the further propagation of corrosion. The analysis of wedge exfoliation that occurred near the A area in Figure 13 b can be simplified into the mechanical model shown in Figure 14 . Under the action of the expansion force of the corrosion product in the A 1 area, structures of aluminum alloy on the upper side (part 1) exfoliated outwardly, and the intergranular corrosion of structures under the lower side (part 2) also occurred along the L direction. Compared to the area of un-corroded substrate, the exfoliation of the structures on both the upper and lower sides of the A 1 area can be considered as the bending of the cantilever beam under load. For the equilibrium of part 1, the wedging force of corrosion products, F 1 , and the bending moment, M 1 , produced at the cantilever beam base, C 1 , must be in balance. Similarly, for the equilibrium of part 2, the wedging forces of corrosion products, F 2 and F 3 , and the bending moment, M 1 , produced at the cantilever beam base, C 2 , must be in balance. It was obvious that before the part 1 had been completely exfoliated outwardly, if part 2 appeared the same degree of wedge exfoliation as part 1, it would require a greater wedging force of the corrosion products in the B area. However, due to the fact that the intergranular corrosion in the B area was not deep enough to provide enough wedge force, part 2 does not form the obvious wedge exfoliation as shown in the A 1 area. Figure 14. Mechanical model of wedge exfoliation analysis. 4. Conclusions (1) After exposure for 20 years in the Wanning test sites, the epoxy coating had been partially destroyed, and the exfoliation corrosion had occurred on the extruded 7075-T6 aluminum alloy substrate. The remaining thicknesses of epoxy coatings for macroscopically intact coating areas followed a normal distribution and decreased linearly. (2) The corrosion resistance of epoxy coatings decreased with the increase of exposure time. After 12 years of exposure, the coating still had a protective effect. After 20 years, although the coating was apparently intact, it had lost its protective characteristics and EIS results showed the occurrence of inductive characteristics. The reason was the formation of cracks between the local coating and the anodic oxide film and of cracks within the coating, which reduced the resistance of the coating to aggressive mediums. (3) The anodic oxide film under the epoxy coating bubbling area had been destroyed and the aluminum alloy substrate had also been corroded. The corrosion products in the interior of the coating bubbling area were mainly hydroxides of aluminum. (4) As the continuous chain of η phase was distributed at the grain boundary, the extruded 7075-T6 aluminum alloy was sensitive to exfoliation corrosion. The propagation of exfoliation corrosion in the S-L section was along the direction of extrusion. Cracks between the lumps of corrosion products provided the channels for the transmission of corrosion mediums, which created conditions for the further propagation of corrosion. Moreover, the structures of the aluminum alloy on the upper side of the corrosion product area displayed a more evident exfoliation than the lower side. 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[ Google Scholar ] Figure 1. Schematic drawing of the specimen’s test section. Figure 2. Location of the exposure test site. Figure 3. Cross-sections of specimens exposed for different years (one side of the upper surface coatings): ( a ) 7 years; ( b ) 12 years; ( c ) 20 years. Figure 4. Surface appearance of coated test specimens exposed to the Wanning test site: ( a ) 7 years; ( b ) 12 years; ( c ) 20 years; ( d ) 20 years (whole upper surface). Figure 5. XRD spectrum of exfoliation corrosion products. Figure 6. Result of the probability paper test of normal distribution (20 years’ data from coating-still-existing areas). Figure 7. Relationship between atmospheric corrosion exposure years and remaining thickness (20 years’ data from coating-still-existing areas). Figure 8. EIS results and its fitted data of coated specimens exposed for 7, 12, and 20 years: ( a ) Bode log \|Z\| versus log (f/Hz) plots; ( b ) Bode phase angle versus log (f/Hz) plots. Figure 9. Electrochemical equivalent circuits for fitting the impedance data: ( a ) 7 and 12 years; ( b ) 20 years. Figure 10. Backscattered electron image of the S-L section of the specimen exposed for 20 years: ( a ) SEM morphology; ( b ) local enlarged SEM morphology. Figure 11. S-L sectional analysis in the coating bubbling area of coated specimen exposed for 20 years: ( a ) SEM morphology; ( b ) EDS spectrum of the corrosion products; ( c – f ) the elemental distributions of O, Al, S and C obtained by EPMA. Figure 12. 7075-T6 aluminum alloy ( a ) S-L metallographic section; ( b ) grain boundary structure and EDS analysis taken by TEM. Figure 13. S-L sectional analysis in the junction area between the corroded and un-corroded substrate of the specimen exposed for 20 years: ( a ) SEM morphology; ( b ) local enlarged SEM morphology; ( c ) EDS spectrum in the lump area of the corrosion products; ( d ) EDS spectrum in the cracking area of the corrosion products. Figure 14. Mechanical model of wedge exfoliation analysis. Table 1. Chemical composition (wt %) of extruded 7075-T6. Element Si Fe Cu Mn Mg Cr Zn Ti Al Weight fraction (%) 0.50 0.50 1.68 0.39 2.31 0.18 6.01 0.10 Bal. Table 2. Average environmental data measured in 1997 at Wanning. Environmental Characteristics Average (Ranges) Air temperature(°C) 23.9 (31.4–17.8) Relative humidity (%) 87.6 (100.0–80.0) Velocity of wind (m/s) 2.4 SO 2 (mg/m 3 ) 0.0452 NO 2 (mg/m 3 ) 0.0020 Cl − deposition rate (mg/(m 2 d)) 14.5875 Table 3. Distribution parameters and correlation coefficients of the distribution models. Years Normal Log-normal Logistic Weibull Gumbel μ σ r μ σ r μ σ r σ β r μ σ r 7 39.5 4.0 0.993 1.6 0 0.989 39.5 2.3 0.985 41.2 12.0 0.985 37.6 3.3 −0.955 12 31.9 3.4 0.993 1.5 0 0.991 31.9 1.9 0.990 33.3 11.7 0.983 30.4 2.7 −0.968 20 22.5 2.5 0.986 1.4 0.1 0.972 22.5 1.4 0.985 23.5 11.1 0.995 21.3 2.1 −0.935 Table 4. Simulated parameters of EIS results of coated samples exposed for 7, 12, and 20 years. Samples Q c (F cm −2 ) n c R c (Ω cm 2 ) Q f (F cm −2 ) n f R f (Ω cm 2 ) C dl (F cm −2 ) R t (Ω cm 2 ) R l (Ω cm 2 ) L (H) Chi-Square Value ( χ 2 ) 7 years 1.01 × 10 −9 0.90 6.83 × 10 5 1.24 × 10 −8 0.74 2.41 × 10 7 2.57 × 10 −9 4.61 × 10 7 2.466 × 10 −3 12 years 2.64 × 10 −8 0.75 8.62 × 10 4 4.61 × 10 −8 0.81 4.95 × 10 6 8.62 × 10 −8 5.38 × 10 6 1.066 × 10 −3 20 years 9.51 × 10 −7 0.59 7.08 × 10 3 2.10 × 10 −7 0.89 1.41 × 10 4 2.05 × 10 −7 2.49 × 10 5 6.99 × 10 4 9.97 × 10 4 1.134 × 10 −3 © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/ ). Zhang, S.; He, Y.; Zhang, T.; Wang, G.; Du, X. Long-Term Atmospheric Corrosion Behavior of Epoxy Prime Coated Aluminum Alloy 7075-T6 in Coastal Environment. Materials 2018, 11, 965. https://doi.org/10.3390/ma11060965 AMA Style Zhang S, He Y, Zhang T, Wang G, Du X. Long-Term Atmospheric Corrosion Behavior of Epoxy Prime Coated Aluminum Alloy 7075-T6 in Coastal Environment. Materials. 2018; 11(6):965. https://doi.org/10.3390/ma11060965 Chicago/Turabian Style Zhang, Sheng, Yuting He, Teng Zhang, Guirong Wang, and Xu Du. 2018. "Long-Term Atmospheric Corrosion Behavior of Epoxy Prime Coated Aluminum Alloy 7075-T6 in Coastal Environment" Materials11, no. 6: 965. https://doi.org/10.3390/ma11060965 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Article Metrics	https://www.mdpi.com/1996-1944/11/6/965
Debate: Referendum on Scottish Independence - 13th Nov 2017 - Ged Killen extracts Mon 13th Nov 2017 - Commons - Referendum on Scottish Independence debate Ged Killen contributions to the 13th November 2017 Referendum on Scottish Independence debate Referendum on Scottish Independence Debate Full Debate: Read Full Debate Department: Scotland Office Referendum on Scottish Independence Ged Killen Excerpts Monday 13th November 2017 (5 years, 7 months ago) Westminster Hall Gerard Killen (Rutherglen and Hamilton West) (Lab/Co-op)- Hansard- Copy Link- Will the hon. Gentleman give way? Mr Jack- Hansard- Copy Link-- Excerpts I shall make some progress first. That message resonated with Scots right across the country, quite simply because people are fed up with Nicola Sturgeon and the SNP putting their obsession with our constitution ahead of governing in the interests of Scotland. Today’s debate is not about whether Scottish independence would be a good thing; I argue strongly that it would not be. It is another debate about process, which, as the hon. Member for Linlithgow and East Falkirk (Martyn Day) made clear, is something that the SNP loves. Constantly talking about this issue is like having Cicero’s sword of Damocles looming over the Scottish economy. --- Later in debate --- The Secretary of State for Scotland (David Mundell)- Hansard- Copy Link-- Excerpts I am delighted to serve under your chairmanship, Sir Roger, and I commend Mr Bailey for his earlier efforts in chairing this debate, which opens for many people outwith Scotland a window on Scottish politics. When I considered replying to this debate, I was, like the hon. Member for Caithness, Sutherland and Easter Ross (Jamie Stone), mindful of the words of The National, which indicated this debate was very important. It is therefore surprising that less than half of the SNP Members of Parliament sought to even attend the debate, never mind take part. Let us make that absolutely clear, so that it is on the record, before we hear about the next Unionist conspiracy to make sure that only two or three SNP MPs got to speak, while Unionists crowded them out. It was a choice not to take part in this debate, which I think readers of The Nationalwill be most disappointed to hear. We have added in some ways to the collection of human knowledge. It is disappointing that the Westminster leader of the party, the right hon. Member for Ross, Skye and Lochaber (Ian Blackford), has left. I am sure the hon. Member for Kilmarnock and Loudoun (Alan Brown) said one thing that he would have agreed with: anything Alex Salmond says cannot be relied on. It is good to get that on the record before that well-known Bolshevik begins his new career. One point that will be of interest to many yes voters and SNP voters is that the position of the SNP is to block the UK leaving the EU. That will not go down well with the 500,000 yes voters and the 400,000 SNP voters. I do not think it will go down well with Jim Sillars; I look forward to hearing his response. It will not go down well with Alex Neil and the SNP MSPs who voted to leave the EU, but at least the position is clear: the SNP is for blocking the UK leaving the EU. Another point flushed out, which was clear from several Members and certainly clear in the speech made by the hon. Member for Edinburgh East (Tommy Sheppard), is that another independence referendum, in the view of the SNP, is simply paused. It is not over, not stopped, but paused. That is why there is a very important message to everyone listening to this debate. Every vote ever cast for the SNP will be taken as a vote in support of another independence referendum and in support of independence. That is the case. We have heard it justified as to why the SNP is entitled to take this position, because every single person who voted for them wanted another independence referendum and wants independence. So Scotland beware: vote SNP, get another independence referendum. We have to be very clear on that. From a Unionist point of view, we could take some solace in the complacency of the SNP—something the hon. Member for East Dunbartonshire (Jo Swinson) referred to. The fact that the SNP came within 600 votes of losing another six seats does not seem to have been taken on board. Earlier, the hon. Member for Argyll and Bute (Brendan O'Hara) was promoting the support for the SNP in his constituency, but forgot to tell us that his vote came down by nearly 10% and the Conservative vote went up by 18%. That was a clear message from his voters that they did not want to hear about independence. I thought that the hon. Member for Linlithgow and East Falkirk (Martyn Day) sought to offer a balanced view of the two petitions; he mentioned both, and that probably fulfilled his obligation. What he did not say, of course, was that the petition asking for an independence referendum only got heard on the back of the 220,000 people who did not want an independence referendum, because a petition that attracted fewer than 40,000 votes would not in itself get a debate in Parliament. We have had an independence referendum—that was the theme of much of what has been said. It was a legal and fair referendum. Many aspects of the engagement were welcome. In particular, I found the school debates in which I took part encouraging, in terms of how our young people applied themselves. Nobody, however, can deny that there were many aspects of that referendum that were seriously unpleasant and that we would not want to hold up as a model. It is important for us in the political class to recognise that although we might go on about how great it was that 84.7% of people voted and all the meetings that were held, ordinary members of the public did not enjoy the referendum process. Other than those who are diehards on both sides of the debate, I do not find people on the doorstep who say, “That 2014 referendum was great—the best time of my life.” What they say—even those who voted yes—is: “I don’t want to go through that again.” Gerard Killen- Hansard- Copy Link- Does the right hon. Gentleman agree that if we were to accept demands for a second independence referendum from the Scottish National party and it was successful in that referendum, it would set a precedent to revisit that decision in a third referendum for Scotland to go back into the United Kingdom? What precedent would that set for the future constitution of the UK? David Mundell- Hansard- Copy Link-- Excerpts The hon. Gentleman is correct. We were told in the Edinburgh agreement that the result would be respected on both sides. The hon. Member for East Lothian (Martin Whitfield) referred to the reconciliation service, at which I was present. I was hopeful, at that point, that it would lead to a way forward. That did not happen. There was a point at which the SNP, and those people who had argued for yes, came out and said that to make their case they needed to make a bigger tent, bring more people in, and convince people. Today, however, we have heard what has become the core of their message: the people of Scotland were duped and we need to do it all over again. That is essentially what we have heard from SNP Members. In the tirade of negativity from the hon. Member for Kilmarnock and Loudoun, nothingpositive was said about what an independent Scotland would be like or would do. In particular, nothing was said about Andrew Wilson’s report on how the £14 billion deficit would be managed. That is a piece of information that I would want, as a Scottish voter, before there was any prospect of opening up another independence referendum. A lot of the arguments have been well rehearsed. I will not respond to the essentially political points made by the shadow Secretary of State for Scotland, the hon. Member for Kirkcaldy and Cowdenbeath (Lesley Laird). We see in Scotland how the Scottish Labour party talks about the Labour party, and that is what she has replicated here today. All of us who support the United Kingdom should follow the example of her colleague, the hon. Member for Coatbridge, Chryston and Bellshill (Hugh Gaffney), who made the case, albeit from a socialist perspective, for the United Kingdom, as my hon. Friend the Member for East Renfrewshire (Paul Masterton) did very eloquently too. The message from this debate, this petition and everything we have heard from the SNP is that we cannot be complacent. We must make the case for the United Kingdom all the time, and ensure that in elections the SNP does not get itself into a position where it can take forward another independence referendum.	https://www.parallelparliament.co.uk/mp/ged-killen/debate/2017-11-13/commons/westminster-hall/referendum-on-scottish-independence
(PDF) Biotransformation of the Proteogenic Amino Acids Phenylalanine, Tyrosine and Tryptophan by Yarrowia Species: An Application to the Preparative Synthesis of Natural Phenylacetic Acid PDF \| The biotransformation of the aromatic amino acids phenylalanine, tyrosine and tryptophan originates a number of bioactive compounds. Yeasts are... \| Find, read and cite all the research you need on ResearchGate Biotransformation of the Proteogenic Amino Acids Phenylalanine, Tyrosine and Tryptophan by Yarrowia Species: An Application to the Preparative Synthesis of Natural Phenylacetic Acid DOI: 10.3390/catal12121638 Authors: Abstract and Figures The biotransformation of the aromatic amino acids phenylalanine, tyrosine and tryptophan originates a number of bioactive compounds. Yeasts are the most used microorganisms for the transformation of (L)-phenylalanine into the flavour phenylethanol. Here, we reported a study on the biotransformation of the proteogenic aminoacids phenylalanine, tyrosine and tryptophan by yeast strains belonging to Yarrowia genus. We found that the latter microorganisms, in high aerobic conditions, metabolise the aromatic amino acids (L)-phenylalanine and (L)-tyrosine with the almost exclusive formation of phenylacetic acid and 4-hydroxyphenylacetic acid, respectively. Differently, the biotransformation of (L)-tryptophan with Y. lipolytica, gave anthranilic acid as the main product. As stated by the European and USA legislations concerning natural flavour production, phenylacetic acid obtained by microbial conversion of phenylalanine of natural origin can be commercialised as a natural flavour. Accordingly, our findings were exploited in a new process, based on the Yarrowia strains-mediated biotransformation of natural (L)-phenylalanine, that allows the large-scale preparation of the high-value, natural flavour, phenylacetic acid. Reference standards used for GC-MS analysis of the biotransformation experiments. … Biotransformation of (L)-phenylalanine with long contact time and high aeration. … Synthesis of PAA acid by biotransformation of (L)-phenylalanine using different Yarrowia strains. … Biotransformation of (L)-phenylalanine, (L)-tyrosine and (L)-tryptophan using Yarrowia lipolytica (DSM 8218). … Citation: Serra, S.; Castagna, A.; Marzorati, S.; Valentino, M. Biotransformation of the Proteogenic Amino Acids Phenylalanine, Tyrosine and Tryptophan by Y arrowia Species: An Application to the Preparative Synthesis of Natural Phenylacetic Acid. Catalysts 2022 , 12 , 1638. https://doi.org/10.3390/ catal12121638 Academic Editor: Gloria Fernandez-Lorente Received: 8 November 2022 Accepted: 6 December 2022 Published: 14 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). catalysts Article Biotransformation of the Proteogenic Amino Acids Phenylalanine, T yrosine and T ryptophan by Y arrowia Species: An Application to the Preparative Synthesis of Natural Phenylacetic Acid Stefano Serra * , Antonio Castagna, Stefano Marzorati and Mattia V alentino Consiglio Nazionale delle Ricerche (C.N.R.), Istituto di Scienze e T ecnologie Chimiche, Via Mancinelli 7, 20131 Milano, Italy * Correspondence: stefano.serra@cnr.it; Tel.: +39-02-2399-3076 Abstract: The biotransformation of the aromatic amino acids phenylalanine, tyrosine and tryptophan originates a number of bioactive compounds. Y easts are the most used microorganisms for the transformation of (L)-phenylalanine into the ﬂavour phenylethanol. Here, we reported a study on the biotransformation of the proteogenic aminoacids phenylalanine, tyrosine and tryptophan by yeast strains belonging to Y arrowia genus. W e found that the latter microorganisms, in high aerobic conditions, metabolise the aromatic amino acids (L)-phenylalanine and (L)-tyrosine with the almost exclusive formation of phenylacetic acid and 4-hydroxyphenylacetic acid, respectively . Differently , the biotransformation of (L)-tryptophan with Y . lipolytica , gave anthranilic acid as the main product. As stated by the European and USA legislations concerning natural ﬂavour production, phenylacetic acid obtained by microbial conversion of phenylalanine of natural origin can be commercialised as a natural ﬂavour. Accordingly , our ﬁndings were exploited in a new process, based on the Y arrowia strains-mediated biotransformation of natural (L)-phenylalanine, that allows the large-scale preparation of the high-value, natural ﬂavour , phenylacetic acid. Keywords: phenylacetic acid; natural ﬂavours; biotransformation; Y arrowia ; biocatalysis; whole-cell process; phenylalanine; tyrosine; tryptophan; anthranilic acid 1. Introduction The Ehrlich pathway [ 1 ] is the most relevant process of amino acid transformation in yeast, whose catabolism is an important source of bioactive compounds. In particular, the aromatic amino acids phenylalanine, tyrosine and tryptophan are precursors of dif ferent classes of compounds of commercial relevance, such as ﬂavours [ 2 – 7 ], phytohormones [ 8 , 9 ], and anti-inﬂammatory , antiviral or antimicrobial agents [ 10 – 12 ]. According to the Ehrlich pathway , after the initial transamination reaction (Figure 1 ), the amino acids 1a – c give the corresponding substituted pyruvic acids 2a – c , which can be the subject of different biochemical transformations. The reduction of the keto functional group affords aryl lactic acids 3a – c whereas the decarboxylation reaction leads to the formation of the aldehydes 4a – c . The latter compounds, usually do not accumulate in yeast cells and are subjected to either reduction or oxidation to afford 2-aryl ethanol 5a – c or 2-aryl acetic acids 6a – c . Phenylalanine transformation is the most important method of production of natural phenylethanol (PE) 5a [ 2 ]. This compound, as well as its derivatives, are widely used as a ﬂavour ingredient [ 13 ] ([ 14 ], p. 1633) and have been recognised as safe for food ﬂavouring [ 15 ], as certiﬁed by the Flavor & Extract Manufacturers Association (FEMA) that included phenethyl alcohol, aldehyde, acid, and related acetals and esters in the list of the substances Generally Recognized As Safe (GRAS). In addition, phenylalanine is currently Catalysts 2022 , 12 , 1638. https://doi.org/10.3390/catal12121638 https://www.mdpi.com/journal/catalysts Catalysts 2022 , 12 , 1638 2 of 13 used as starting material for the synthesis of natural phenylacetic acid (P AA) 6a . Indeed, although the direct microbial biotransformation of 1a into 6a has been exploited in only a few processes [ 3 , 4 ], the main part of the natural PAA is curr ently produced by a two steps pathway , based on the transformation of 1a into PE 5a followed by its oxidation to the corresponding acid by fermentation with Gluconobacter oxidans [ 5 – 7 ]. Catalysts 2022 , 12 , x FOR PEER REVIEW 2 of 15 [ 1 5 ] , as certi f ied by the Fl avor & Extra c t Ma n u facturer s Assoc i ation (FEM A) that include d phenet hyl a l cohol, ald e h y de, acid , a n d rel a t e d a c et als and e s t e rs in t h e list o f t h e subst a nc es G e nera lly R e co gniz ed As Sa f e (G R AS ) . In add i t i on, phe n yla l anine is current ly used as st art i ng m a t e ri al f o r t h e synt he sis o f nat u ra l phenyla c et ic acid (P AA) 6a . Indee d , although the direct microb ial biotran s fo rmation of 1a int o 6a h a s be en exploited in only a few proces ses [3 ,4 ], t h e ma i n part of t h e n a t u ra l PAA is cu rrently produ c ed by a two steps pa thwa y, b a sed on the transf orm a ti on of 1a int o P E 5a followed by its oxid ation to th e correspondi ng a c i d by f e rmenta ti on with Gluconobacter o x ida n s [ 5–7 ]. Figure 1. The biotransformation of the amino acids (L)-phenylalanine 1a , (L)-tyrosine 1b and ( L )- tryptophan 1c through Ehrlich pathway. As desc ribed for alcohol 5a , a l so PAA is a n i m porta n t f l a v our ( pp. 13 61– 136 2, 1656 – 16 5 7 , [ 1 4] ) t h at is u s ed in f oods and b e v e ra ge s for its sweet, hon e y-like odo u r. T h erefore , a n u m b er o f b i ocat alyt i c s y nt heses o f b o t h l a t t e r com p ounds and of t h eir der i v a t i v e s have been develop e d so f a r, incl udin g t h ose b a sed on met a bolic en ginee r ing [1 6 – 2 2 ] . All the proce sses de scribe d above fu lfil th e r e quir em ents of the E u ropean [23] and USA [24] legislation s conc erning n a tural fl avour production. Ind eed, both regulations esta bli s h tha t the bi otra nsf o rma t i o n of a na tur a l precurso r is a ‘natur al me thod’ of synthesis [25] and the flavours obt a ined by these me ans possess the ‘natur al’ status. Since the flavo u rs possess ing th e ‘natural’ status ar e us uall y hundre ds o f times as exp e nsive as thei r synthetic counterp a r ts, a n y new procedur e that provides these compound s in their high-v al ue fo rm can be ve r y prof it ab le. This con s id er at ion is cert ai nly pe rt inent for PAA , whose commercial v a lue range from less than on e hundred euros for the synthetic compound to more tha n a thousa nd euro s for t h e f l avour o f n a t u r a l origin . We h a v e alr e ady dev e lop e d an a l yt ic m e t h ods of origi n a u thenti ca ti on of the mos t relev a nt nat u ral phenylpr opanoid s , inc l ud ing PAA , by mean s o f t h eir st ab le isot ope char act e ris a t i on [ 2 6 – 3 0 ] . I n ad dit i on , we hav e b e e n recent l y in v o lv ed in a rese arch progra m aimed to develop new bioca t alytic sy ntheses of h i gh-v alue na tu ra l fl a v ou rs [3 1– 34 ]. Takin g adv a ntage of o u r acquir ed ex perience, we decided to study the ye asts- mediated transform a tion of the most relevant p r ot eo genic arom at i c am ino acid s , nam e ly p h enyla l anin e, t y ro sine an d t r yp t o p h an . Ou r p r e lim i n ary res u lt s i n dicat e d a re m a rk ab le divers ity am ong ye ast s p ecies and we first d i sc overed that different y e ast strains belongin g t o t h e gen u s Yarrowia a r e ab le t o t r ans f orm d i rect ly p h eny l al anin e 1a int o PAA . Figure 1. The biotransformation of the amino acids (L)-phenylalanine 1a , (L)-tyrosine 1b and (L)-tryptophan 1c through Ehrlich pathway . As de scr ibed f or alc oho l 5a , als o P AA is a n impo rtan t fla vour ( pp. 1361 –136 2, 165 6–1 657 , [ 14 ]) that is used in foods and beverages for its sweet, honey-like odour. Therefore, a number of biocatalytic syntheses of both latter compounds and of their derivatives have been developed so far, including those based on metabolic engineering [ 16 – 22 ]. All the processes described above fulﬁl the requirements of the European [ 23 ] and USA [ 24 ] legislations concerning natural ﬂavour production. Indeed, both regulations establish that the biotransformation of a natural precursor is a ‘natural method’ of syn- thesis [ 25 ] and the ﬂavours obtained by these means possess the ‘natural’ status. Since the ﬂavours possessing the ‘natural’ status are usually hundreds of times as expensive as their synthetic counterparts, any new procedure that provides these compounds in their high-value form can be very proﬁtable. This consideration is certainly pertinent for P AA, whose commercial value range from less than one hundred euros for the synthetic compound to more than a thousand euros for the ﬂavour of natural origin. W e have already developed analytic methods of origin authentication of the most relevant natural phenylpropanoids, including P AA, by means of their stable isotope char- acterisation [ 26 – 30 ]. In addition, we have been r ecently involved in a research program aimed to develop new biocatalytic syntheses of high-value natural ﬂavours [ 31 – 34 ]. T aking advantage of our acquired experience, we decided to study the yeasts-mediated transformation of the most relevant proteogenic aromatic amino acids, namely phenylala- nine, tyrosine and tryptophan. Our preliminary results indicated a remarkable diversity among yeast species and we ﬁrst discovered that different yeast strains belonging to the genus Y arrowia are able to transform directly phenylalanine 1a into P AA. Hence, we exploited these ﬁndings and devised a new preparative process for the 35 ]. Afterward, we extended our research by studying the activity of Y arrowia lipolytica in the biodegradation of both racemic and (D)-phenylalanine, as well as in the transformation of tyrosine and tryptophan. W e reported here the results of these investigations, which point to the great potential of Y arrowia strains in aromatic amino acids transformation. Catalysts 2022 , 12 , 1638 3 of 13 2. Results and Discussion As mentioned before, at ﬁrst we investigated the yeast-mediated transformation of (L)-phenylalanine into PE and P AA. Since the main application of these compounds is their use as ﬂavours in foods and beverages, we selected yeast strains belonging to biosafety level 1, with a strong preference for those recognised with technologically beneﬁcial use in foods [ 36 ]. Accordingly, we set up a preliminary scr eening experiment in which four different yeast strains are fermented in the universal medium for yeasts (YM) containing (L)-phenylalanine (5 g/L), in an aerated ﬂask (T able 1 ). T able 1. Biotransformation of (L)-phenylalanine with short contact time and low aeration. Entry 1 Yeast PE/PAA Ratio PAA (g/L) 6 PE + PAA Yield (%) 7 2 Saccharomyces boulardi 2,3 97/3 0.013 13.1 3 Pichia pastoris 2 97/3 0.013 14.8 4 Yarr owia lipolytica (DSM 8218) 2 21/79 0.470 16.1 5 Yarr owia lipolytica (DSM 8218) 4,5 <1/99 1.675 40.0 1 Reaction conditions: YM containing 5 g/L of (L)-phenylalanine, at 26 ◦ C for 3 days, under shaking (110 rpm). 2 Low aeration conditions: 100 mL of medium in 250 mL Erlenmeyer ﬂask sealed with a cellulose plug. 3 The probiotic strain Saccharomyces boulardi I-3799 was grown at 36 ◦ C. 4 High aeration conditions: 100 mL of medium in 1 L Erlenmeyer ﬂask sealed with a cellulose plug. 5 5 Days of fermentation. 6 Concentration of PAA in the 7 Molar yield of PE + P AA formation. The concentrations of PE and P AA in the fermentation broth were measured by GC-MS analysis, using 2-phenyl-1-propanol and 2-phenylpropanoic acid, respectively , as internal standards. The rat ionale behi nd the choic e of the four str ains lies in t he fact that Sa ccharom yces cere - visiae , the main yeas t present i n baker’s yeast, has b een histor ically used f or PE produ ction [ 1 , 2 ] and thu s can be regar ded as a ‘pos itive cont rol’ in scr eening exp eriments . Sacchar omyces boul ardi is genet ically cor related t o S. cerevis iae , but thi s probioti c microor ganism [ 37 ] can gr ow at a higher t emperatu re (36 ◦ C), allo wing to inve stigate th e biotransf ormation o f phenylala nine at a temper ature rat her unusua l for yeasts. Pichia pastoris [ 38 ] is used in the food industry , as a model organism for biological studies and as a system for the expression of cloned genes. Even more interesting is the case ] and has been included in the list of GRAS microorganisms. This yeast is classiﬁed as oleaginous [ 40 ] as it is able to accumulate intracellularly a considerable amount of lipids. It has found a number of applications in industry and some strains have been employed for the transformation of phenylalanine into PE [ 41 , 42 ]. Our biot ransform ation expe riments hig hlighted t hat S. cerev isiae , S. bo ulardi and P . pastoris transf ormed (L)- phenylal anine in PE (en tries 1–3) w ith high sele ctivity si nce the PE/P AA ratio was supe rior to 97/3 f or all cases . On the contra ry , Y . lipolyti ca produce d mainly P AA (en try 4) as the mea sured PE/ PA A ratio was 21 /79. The latter dat a disagre e with those r eported in t he litera ture [ 41 , 42 ] openin g the questi on about the i nfluence of t he experim ental condi tions and of the str ain variab ility on the ye ast activi ty . As the P AA formation is subject to an oxidation reaction, we set a ﬁfth experiment (entry 5) in which the biotransformation was performed with higher aeration conditions and with longer contact time (5 days instead of 3). The results of the latter trial were remarkable, indicating the importance of the oxygen supply for Y . lipolytica activity . Almost only P AA was formed, whereas the overall yield increased from 16.1% (entry 4) to 40.0%. experi ments that e xamined a la rger selec tion of yeas t strains, wh ose activi ties, in high a eration condit ions, wer e compare d with that of th e Y. l ipolytica s train used b efore (DS M 8218). Besides the four microorganisms described in T able 1 , we singled out other oleaginous yeasts, belonging either to Ascomycota or to Basidiomycota division. In this context, we utilised Candida boidinii , Starmerella bombicola , Debaryomyces hansenii , Cryptococcus curva- tus , Sporidiobolus johnsonii, and Phafﬁa rhodozyma, which have been largely employed in industrial processes [ 40 , 43 – 46 ] for the preparation of a number of food additives including Catalysts 2022 , 12 , 1638 4 of 13 ﬂavours and dietary supplements. Finally , within non-oleaginous yeasts, we selected two important microorganisms largely used in food transformation, namely T orulaspora delbrueckii [ 47 ] and Kluyveromyces marxianus [ 48 ]. Accordingly , the biotransformation experiments were performed using a starting concentration of phenylalanine of 4.5 g/L, high aeration conditions, and a rather long contact time (7 days). The results are collected in T able 2 and allow some important comments. Firstly, we can observe that the new biotransformation conditions do not affect the PE/P AA ratio only for Saccharomyces strains (entries 1 and 2) where PE remain almost the exclusive product. Otherwise, there is high variability in PAA pr oductivity for all other strains tested. Debaryomyces hansenii and Torulaspora delbrueckii (entries 7 and 8) transform phenylalanine mainly into PE. Pichia pastoris and Kluyveromyces marxianus (entries 3 and 9) produced more PE than P AA, Candida boidinii and Phafﬁa rhodozyma (entries 5 and 12) produced more P AA than PE, whereas Y arrowia lipolytica and Starmerella bombicola (entries 4 and 6) produce almost exclusively P AA. T able 2. Biotransformation of (L)-phenylalanine with long contact time and high aeration. Entry 1 Yeast PE/PAA Ratio PAA (g/L) 4 PE + PAA Yield (%) 5 1 Saccharomyces cerevisiae 97/3 0.02 16.8 2 Saccharomyces boulardii 2 96/4 <0.01 3.7 3 Pichia pastoris 52/48 0.41 24.4 4 Yarr owia lipolytica (DSM 8218) <1/99 1.97 53.0 5 Candida boidinii 35/65 0.87 37.9 6 Starmerella bombicola <1/99 0.05 1.3 7 Debaryomyces hansenii 88/12 0.01 3.0 8 T orulaspora delbrueckii 87/13 0.06 13.4 9 Kluyveromyces marxianus 61/39 0.61 45.1 10 Cryptococcus curvatus 3 3 - 11 Sporidiobolus johnsonii 3 3 - 12 Phafﬁa rhodozyma 39/61 0.02 0.9 1 Reaction conditions: YM containing 4.5 g/L of (L)-phenylalanine, at 26 ◦ C for 7 days, under shaking (140 rpm). High aeration conditions: 100 mL of medium in 1 L Erlenmeyer ﬂask sealed with a cellulose plug. 2 The probiotic strain Saccharomyces boulardi I-3799 was grown at 36 ◦ C. 3 PE and PAA ar e not present in the broth after 7 days of fermentation. 4 Concentration of PAA in the fermentation br oth after 7 days, measured by GC-MS analysis, using 2-phenylpropanoic acid as internal standard. 5 Molar yield of PE + PAA formation. The concentrations of PE and PAA in the fermentation br oth were measured by GC-MS analysis, using 2-phenyl-1-propanol and 2-phenylpropanoic acid, respectively , as internal standards. Besides the PE/P AA ratio, we took into account also the overall productivity (PE + PAA yield). Indeed, all Basidiomycota strains (entries 10–12), S. bombicola , and D. hansenii (entries 6 and 7) show very low productivity (up to 3% overall yield) most likely due to the almost complete utilisation of phenylalanine by fungal metabolism. Furthermore, the Ascomycota Saccharomyces strains, P . pastoris , and T . delbrueckii (entries 1–3 and 8) show overall yields that range from low to modest (up to 25%) whereas Y . lipolytica , C. boidinii , and K. marxianus (entries 4, 5, and 9) give the best performance in term of PE + PAA production (up to 53% total yield). Overall, Y . lipolytica (DSM 8218) proved to be the most suitable microorganism for the transformation of (L)-phenylalanine into P AA as it showed a very high chemical selectivity (P AA/PE > 99/1) and good biotransformation yield (53%). After establishing the potential of Y . lipolytica for PAA synthesis, we investigated the tolerance of the same strain to higher substrate concentrations. Indeed, in order to reduce bioreactor volume, industrial syntheses based on fermentation processes should be performed with high substrate concentrations. Therefore, we ran ﬁve biotransformation experiments in which Y . lipolytica (DSM 8218) was fermented using high aeration conditions, a rather long contact time (7 days), and different concentrations of the substrate. The phenylalanine concentration ranged from 4.5 to 25 g/L (T able 3 , entries 1–5). Catalysts 2022 , 12 , 1638 5 of 13 T able 3. Synthesis of phenylacetic acid by biotransformation of (L)-phenylalanine, at different concentrations, using Y arrowia lipolytica (DSM 8218). Entry 1 (L)-Phenylalanine Starting Concentration (g/L) PAA Yield (%) 2 1 4.5 51.0 2 8.5 55.4 3 12.5 67.1 4 18.0 52.6 5 25.0 24.2 1 Reaction conditions: YM containing (L)-phenylalanine, at 28 ◦ C for 7 days, under shaking (140 rpm). High aeration conditions: 100 mL of medium in 1 L Erlenmeyer ﬂask sealed with a cellulose plug. 2 Yields are determined after work-up by isolation of the formed PAA. The experiment performed at the lower substrate concentration (entry 1, 4.5 g/L) correspond to that described in T able 2 , with entry 4. The measured yields are almost identical, indicating a good reproducibility of the experimental procedure. According to the data collected in the table, we observed that the increase of the substrate concentration at ﬁrst corresponded to an increase of the P AA isolated yields (entries 1–3), up to 67.1%. Then, higher phenylalanine concentrations initially gave a slightly lower yield (entry 4) and ﬁnally produced a drastic reduction of the same (entry 5). Overall, we can conclude that Y. lipolytica gives the best P AA yields with a substrate concentration of around 12.5 g/L. Although fermentation is possible even at an initial concentration of 25 g/L, we observed a considerable transformation inhibition, most likely All the above-described experiments have been performed using the same Y . lipolytica strain (DSM 8218). Since the latter microbial strain possesses a speciﬁc ability to oxidise aromatic hydrocarbons [ 49 ], we could not exclude that the aptitude of Y. lipolytica to produce P AA was dependent on strain origin and on its genetic variability . Thus, we decided to investigate whether other Y arrowia strains or species are able to perform the same biotransformation. It is worth noting that since its ﬁrst description [ 50 ], the genus Y arrowia was monotypic, accommodating the single species Y. lipolytica . Only recently , the new molecular techniques of microbial typing allowed the introduction of numerous novel species [ 51 – 55 ]. In order t o study their poten tial use for P AA synthes is, we thus selecte d different sp ecies belongin g to Ya rrowia genus an d one further strai n of Y. l ipolytica . More specifica lly , we singled out a number o f strains that ar e placed in differ ent positions of t he last described Y arrowia clade phyl ogenetic tree [ 55 ] or that wer e isolated fro m very differen t natural envir onments. All the sele cted strains wer e employed in biot ransformatio n experiments wit h high aeration conditio ns, a rather long con tact time (7 days), a nd a phenylalanin e concentration o f 12.5 g/L (T able 4 ). According ly , we ran seven flask f ermentation tri als (entries 1–7) . tank (DSM 8218) and the second one isolated from marzipan (DSM 70562), gave almost identical results regardless of the differ ent isolation substrates, affording P AA in 67 and 69% molar yield, respectively . Similarly, Y . deformans and Y . bubula (entries 3 and 5), which hold well-separated positions in Y arrowia phylogenetic tree, afforded P AA in 64 and 66% molar yield, respectively . Differently , Y. yakushimensis , Y . phangngaensis , and Y. brassicae species (entries 4, 6, and 7), gave disappointing results affording P AA in 24, 27, and 32% yield, respectively . Seen together, these results indicate that neither the strain origin nor the genetic similarity between species can be used as the sole parameter to identify the best P AA producers in Yarr owia clade. Overall, we selected Y . lipolytica , Y. deformans , and Y . bubula as the best Y arrowia species for the production of natural P AA. Accordingly , we used the corresponding four strains (entries 8–11) for the preparative scale synthesis of P AA. To this end, we performed the biotransformations in the bioreactor , using the same experimental conditions employed Catalysts 2022 , 12 , 1638 6 of 13 for ﬂask fermentations. For all strains, we observed a deﬁnite increase in PAA yield, most likely due to the better control of the aeration and of the pH in the bioreactor . T able 4. Synthe sis of P AA acid by biot ransforma tion of (L)-ph enylalani ne using diffe rent Y arrowi a strains. Entry 1 Y arrowia Strain Fermentation Conditions PAA Yield (%) 4 2 Y arrowia lipolytica (DSM 70562) Flask 2 69 3 Y arrowia deformans (DSM 70561) Flask 2 64 4 Y arrowia yakushimensis (CBS 10252) Flask 2 24 5 Y arrowia bubula (CBS 12934) Flask 2 66 6 Y arrowia phangngaensis (CBS 10407) Flask 2 27 7 Y arrowia brassicae (CBS 15225) Flask 2 32 8 Y arrowia lipolytica (DSM 8218) Bioreactor 3 71 9 Y arrowia lipolytica (DSM 70562) Bioreactor 3 75 10 Y arrowia deformans (DSM 70561) Bioreactor 3 72 11 Y arrowia bubula (CBS 12934) Bioreactor 3 68 1 Reaction conditions: YM containing 12.5 g/L of (L)-phenylalanine, at 28 ◦ C for 7 days. 2 Flask fermentation: 100 mL of medium in 1 L Erlenmeyer ﬂask sealed with a cellulose plug, under shaking (140 rpm). fermentation: 5 L fermenter loaded with 2 L of medium. Airﬂow, stirring speed, and pH were set to 1 L/min, 250 rpm, and 6.5, respectively . 4 Yields are determined after work-up by isolation of the formed PAA. Then, all the fi ndings above des cribed, allowed us t o patent [ 35 ] a new prep arative proces s for the synthesis o f natural P AA starting f rom natural (L) -phenylalanin e. Our a p- proach c ompares favou rably with the pr eviously repo rted whole-cell p rocedures b ased on the direct t ransformation o f phenylalanine i nto P AA [ 3 , 4 ]. Indeed, the latte r approaches cl aimed inferior y ields and make use of m icroorgan isms not suitable f or food flavour pr oduction, as those bel onging to BSL 2 [ 3 ]. On the contrary , our pro cess takes advan tage of Y arrowia strains po ssessing the GRAS s tatus and prod ucing natural P AA in isol ated yields rang ing from 70 to 7 5%. Moreover , our meth od is user-frie ndly , the isolation o f the product do es not requir e difficult sep aration proce dures and its com mercial viabi lity is secured b y the high differ ence in value betwe en the starting ma terial, namely nat ural (L)-pheny lalanine (less th an 200 euro /kg), and the pro duced natural P AA (mor e than 1000 euro /kg). A further extension of our research was the study of the transformation of the amino acid phenylalanine in relation to its enantiomeric composition. Since the ﬁrst step in the Ehrlich pathway is the transamination reaction, which is catalysed by different transam- inases, the yeast could not able to transform the unnatural (D)-enantiomer. Indeed, the latter enzymes usually catalyse the preferential transformation of (L)-phenylalanine with high enantioselectivity . Thus, we arranged a new set of biotransformation trials that employed the same experimental conditions and different substrate compositions. Accordingly , either (L)-, (DL)- and (D)-enantiomers of phenylalanine, in the universal medium for yeasts (YM) and with a starting concentration of 10 g/L, were fermented using Y . lipolytica (Table 5 , entry 1–3). As previously described, (L)-phenylalanine was converted into PAA (entry 1) in good yield (53.4%) whereas the transformation of the racemic amino acid (entry 2) was more difﬁcult, affording P AA in only 25.5% yield. These data could be explained assuming that Y . lipolytica is either unable to transform the (D)-isomer or the transformation rate of the latter enantiomer is much inferior to that of (L)-isomer. T o investigate better this point, we examined the biotransformation experiments in which only (D)-phenylalanine was present in the fermentation broth. We observed that also in this case (entry 3) P AA was formed, even if in a lower amount (12.1%). Since yeast could able to uptake (L)-phenylalanine from the proteins present in the medium, we repeated the last experiment using a protein-fr ee medium (PF-YM), which contain neither proteins nor (L)-phenylalanine. As a result, PAA was yet produced although the isolated yield was further reduced to 5.5% (entry 4). Catalysts 2022 , 12 , 1638 7 of 13 T able 5. Synthesis of phenylacetic acid by biotransformation of (L)-, (DL)-, and (D)-phenylalanine, using Y arrowia lipolytica (DSM 8218). Entry 1 Phenylalanine Enantiomer Fermentation Medium PAA Yield (%) 2 1 (L) YM 53.4 2 (DL) YM 25.5 3 (D) YM 12.1 4 (D) PF-YM 5.5 1 Reaction conditions: YM or PF-YM containing 10 g/L of phenylalanine, at 28 ◦ C for 7 days, under shaking (140 rpm). High aeration conditions: 100 mL of medium in 1 L Erlenmeyer ﬂask sealed with a cellulose plug. 2 Yields are determined after work-up by isolation of the formed P AA. Overall, these results demonstrated that Y. lipolytica pr eferentially converted (L)- phenylalanine into P AA whereas the transformation of the (D)-enantiomer was very slow. This behaviour could be explained either by invoking the production of different transami- nases during Y arrowia fermentation or by considering the possibility of a slow process of amino acid racemisation within microbial catabolism of the same. As a ﬁnal point, we investigated the potential of Y . lipolytica in the transformation of two further relevant proteogenic aromatic amino acids, namely tyrosine and tryptophan. To this end, we devised three experiments in which the same Y . lipolytica strain was fermented in presence of either (L)-phenylalanine, (L)-tyrosine, or (L)-tryptophan, employing the same experimental conditions. In order to use the same amino acid concentration for all trials, we set a value of 5 g/L for each trial, which was the highest possible concentration in YM for the less soluble amino acid tyrosine. Accordingly , the results of the biotransformation trials showed very interesting re- sults (T able 6 ). T able 6. Biotransformation of (L)-phenylalanine, (L)-tyrosine and (L)-tryptophan using Y arrowia lipolytica (DSM 8218). Entry 1 Amino Acid Compounds 2–6 2 Other 2 1 (L)-Phenyl alanine 6a (58.3%) - 2 (L)-T yrosine 6b (47.7%) - 3 (L)-Tryptophan 3c (16.0%); 6c (1.3%) Anthranilic acid (33.6%) 1 Biotransformation conditions: 200 mL of YM medium containing 5.0 g/L of the suitable amino acid, at 26 ◦ C for 7 days, under shaking (140 rpm), in 1 L Erlenmeyer ﬂask sealed with a cellulose plug. 2 The compounds were identiﬁed by GC-MS analysis. Molar yields (%) are determined after work-up by chromatographic isolation of the products. First, we observed that (L)-phenylalanine and (L)-tyrosine were transformed in the same way affording exclusively the corresponding arylacetic acid, namely P AA 6a and 4-hydroxyphenyl acetic acid 6b , in 58 and 48% yield, respectively (entries 1 and 2). It is worth noting that 4-hydroxyphenyl acetic acid is a relevant bioactive compound possessing antioxidant [ 11 ] and anti-inﬂammatory [ 12 ] properties. Despite this fact, its synthesis from tyrosine by whole cell process has not been reported yet. Only very recently [ 56 ], research on the transformation of lignin-related phenylpropanoids has described the biotransformation of p -cumaric acid into 6b using engineered E. coli . As a ﬁnal point, we observed that Y . lipolytica transformed (L)-tryptophan in a com- pletely different way (entry 3). Indolacetic acid 6c was the minor metabolite, generated 5c nor indole-3-pyruvic acid 2c was formed. Differently , indole-3-lactic acid 3c was produced in signiﬁcant amounts (16% yield) whereas anthranilic acid (2-aminobenzoic acid) turned out to be the major product synthesised from (L)-tryptophan 1c by Y . lipolytica . The latter compound is most likely produced through a different pathway that involves the oxidation of the indole ring, not necessarily related to the Ehrlich pathway . It should be pointed out that both methyl and ethyl esters of the aforementioned acid are relevant ﬂavours Catalysts 2022 , 12 , 1638 8 of 13 ([ 14 ], pp. 569, 1213 –1214). Therefore, the Y. lipolytica mediated transformation of natural tryptophan could represent new access to the latter ﬂavours in natural form. In conclusion, the present study demonstrates that different yeast strains belonging to Y arrowia genus metabolise the aromatic amino acids (L)-phenylalanine and (L)-tyrosine and 4-hydroxyphenylacetic acid 6b , respectively , in good yield. Except for minor differences in terms of yield, both biotransformation processes hold preparative signiﬁcance, as they are easily scalable from ﬂask trial to bior eactor production and the produced arylacetic acids can be isolated from the fermentation broth, in almost pure form, by simple extraction with a suitable solvent. By these means, employing as substrates (L)-phenylalanine of natural origin, the obtained acid can be labelled as natural allowing the production of the high-value ﬂavour phenylacetic acid. On the contrary , the transformation of (L)-tryptophan with Y. lipolytica gave indoleacetic acid 6c in a trace amount. For this biotransformation, anthranilic acid was the main product, alongside a minor amount of indole-3-lactic acid 3c . Since the esters of anthranilic acid are natural ﬂavours, the yeast-mediated transformation of (L)-tryptophan deserves further consideration and will be the subject of our next studies. 3. Materials and Methods 3.1. Materials and General Methods All air and moisture-sensitive reactions were carried out using dry solvents and under a static atmosphere of nitrogen. All solvents and reagents were of commercial quality and were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-Phenylpropanoic acid was prepared by carbonation of (1-phenylethyl)-magnesium The meth yl esters of 2 -phenylpr opanoic a cid 8 , phenyl acetic aci d 12 , 4-hydr oxyphen yl acetic a cid 15 , 3-indo le acetic ac id 18 , phenyl p yruvic ac id 10 , 4-hydr oxyphen yl pyruvic a cid 13 , indole -3-pyru vic acid 16 , 3-phen yllactic a cid 11 , 3-(4-h ydroxyp henyl)la ctic acid 14 , indole - 3-lact ic acid 17 9 (Figur e 2 ) were pr epared b y treatme nt at 0 ◦ C of the corr esponding a cid with an ex cess of an ethe real solu tion of fre shly-pre pared dia zomethan e. Catalysts 2022 , 12 , x FOR PEER REVIEW 9 of 15 3. Ma t e ri als a nd M e th ods 3.1. Ma terials and Gen eral Metho d s All air and m o isture -sen sit i ve re actions were carrie d out using d r y solvents and un- der a st at ic at m o sp here of nit r ogen. Al l solv ent s and reagent s wer e of com m e rc ia l q u al it y and we re p u r c hased from Sigm a- Ald r ic h (St . Lou i s , MO, U S A ) . 2- Phenylp r op anoic ac id w a s p r ep a r ed b y carb on at ion of ( 1 -p henyl e t h yl)-m agne si um bromide, in t u rn, obt a ined by re act i on o f (1-bromoethyl)ben z ene w i th magn esium turn- ing. The methyl e s ters o f 2-phenylpropan oic ac id 8 , phe n ylacetic ac id 12 , 4 - hy droxy - phenyl acetic ac id 15 , 3- in dole acet ic ac id 18 , phenyl pyruv i c a c id 10 , 4-hydrox yphenyl pyruvic a c id 13 , ind o le -3 -p yruvic ac id 16 , 3-phen yl la ct ic ac id 11 , 3- (4 - h ydroxyphenyl )la c - ti c a c id 14 , in dole- 3 -l act i c acid 17 , and ant h ran i l i c ac ids 9 (Fig ure 2) we re prep ared by trea tment a t 0 °C of the correspondi ng a c i d wi th an excess of an etherea l sol u ti on of fresh l y - prepared d i azomet hane. Figure 2. Reference stan dards used for GC-MS analysis of the biot ransfor m ation experiments. 3. 2. Mi croor ga ni sms an d Gro w t h Me di a Sacc harom y ces cerevisiae (Ty p e II YSC2, L o t BCBR1308) was purchased from Sig m a- Aldr ich (St. L o uis , MO , U S A). Sacc harom y ces boulardii (pr o biot ic st ra in SB8 0 ® , I-37 99 ) wa s p u rcha sed f r om AR Fitofarma s.r.l (Assago , Italy). Pich ia p a storis ( Komag a taella p a storis, DSM 70 382 ), Yarrowia lip olytica ( D SM 821 8) , Yar - rowia lip ol y t ica ( D SM 7 056 2) , Yarrowia deformans ( D SM 7 056 1) , Tor u laspora delbruec kii (DS M 7 048 3) , Debar y om yces hanse n i i ( D SM 70 59 0) , Cand id a b o id inii ( D SM 7 002 6) , Starm erella bom- bicola ( D SM 27 465 ), Kluyver o myce s mar x ia nus ( D SM 7007 3) , Ph affia rhod ozyma ( Xa n t ho p hyl - lomyces de ndr o rhous , DMS 56 26) , Cr yptoco ccus c u rva t us (DSM 7 002 2) , a n d Sp ori d iobolus joh n - sonii ( D S M 7 0 5 8 0 ) w e r e p u r c h a s e d f r o m D S M Z G m b H c o l l e c t i o n ( B r a u n s c h w e i g , G e r - ma ny). Yarrowia yak u shime n sis (CBS 102 52) , Yarrowia b u bula ( C BS 1 293 4) , Ya rro w i a pha ngnga e nsis ( C BS 10 407 ), a n d Yarro wia brassicae ( C BS 15 225 ) were purcha sed from the CBS-K N AW collect ion ( U t r echt , The Ne t h erland s) . Figure 2. Reference standards used for GC-MS analysis of the biotransformation experiments. Catalysts 2022 , 12 , 1638 9 of 13 3.2. Microorganisms and Growth Media Saccharomyces cerevisiae (T ype II YSC2, Lot BCBR1308) was purchased from Sigma- Aldrich (St. Louis, MO, USA). Saccharomyces boulardii (probiotic strain SB80 ® , I-3799) was purchased from AR Fito- farma s.r.l (Assago, Italy). Pichia pas toris ( Komagata ella pastoris, DS M 70382), Y arrowia l ipolytica (DSM 82 18), Y arrowia lipolyti ca (DSM 70562), Y arro wia deformans (D SM 70561), T orulasp ora delbruecki i (DSM 70483), Debaryom yces hansenii (DS M 70590), Candid a boidinii (DSM 700 26), Starmere lla bombicola (DSM 27465 ), Kluyveromy ces marxianus (DS M 70073), Phaffi a rhodozyma ( Xa nthophyllomyc es dendro rhous , DMS 5626), Cry ptococcus curva tus (DSM 70022), and Spor idiobolus johns onii (DSM 70580 ) were purcha sed from DSMZ Gmb H collection (Bra unschweig, Germ any). Y arrowia yakushimensis (CBS 10252), Yarr owia bubula (CBS 12934), Yarrowia phangngaensis (CBS 10407), and Yarr owia brassicae (CBS 15225) were purchased from the CBS-KNA W collection (Utrecht, The Netherlands). The biotransformation experiments were performed using two different media, namely a universal medium for yeasts (YM) and a protein-free medium for yeasts (PF-YM), de- pending on the microorganism used. YM composition: yeast extract (3 g/L), malt extract (3 g/L), peptone from soybeans (5 g/L), glucose (10 g/L), trace element solution (10 mL). PF-YM composition: glucose (10 g/L), soluble starch (3 g/L), maltose (2 g/L), yeast nitrogen base (0.7 g/L), trace element solution (10 mL). T race elements solution: FeCl 3 (50 mM), CaCl 2 (20 mM), MnCl 2 (10 mM), ZnSO 4 (10 mM), CoCl 2 (2 mM), CuCl 2 (2 mM), NiCl 2 (2 mM), Na 2 MoO 4 (2 mM), Na 2 SeO 3 (2 mM), H 3 BO 3 (2 mM). 3.3. Biotransformation Experiments and Preparative Synthesis of Phenylacetic Acid 6a and 4-Hydroxy-Phenylacetic Acid 6b All the biotransformation experiments were carried out in triplicate and the results presented in the tables are the media of three experimental data. The experiments were performed either in ﬂasks or in a 5 L fermenter (Biostat A BB-8822000, Sartorius-Stedim, Göttingen, Germany). Phenylalanine, tyrosine, and tryptophan were added to the suitable medium before the sterilisation cycle (121 ◦ C, 20 min). The inoculum of the yeast strains was prepared according to the following procedur e: A small amount of the suitable yeast strain was picked up from a petri dish, suspended in 1 mL of sterile saline, and then inoculated in a 100 mL conical pyrex ﬂask containing 40 mL of YM. The ﬂask was shaken for 3 days at 26 ◦ C and 140 rpm. After this period, 1 mL of this active culture was used to inoculate 40 mL of the fresh medium containing the suitable amino acid. For preparative biotransformations, the whole active culture described above was centrifuged 5 min, (4	https://www.researchgate.net/publication/366274374_Biotransformation_of_the_Proteogenic_Amino_Acids_Phenylalanine_Tyrosine_and_Tryptophan_by_Yarrowia_Species_An_Application_to_the_Preparative_Synthesis_of_Natural_Phenylacetic_Acid
Commissioner Of Income-Tax, Mysore v. Purushotam Gangadhar Bhende. \| Bombay High Court \| Judgment \| Law \| CaseMine Get free access to the complete judgment in Commissioner Of Income-Tax, Mysore v. Purushotam Gangadhar Bhende. on CaseMine. Commissioner Of Income-Tax, Mysore v. Purushotam Gangadhar Bhende. Bombay High Court Dec 17, 1974 Case Information CITATION CODES citation codes CASE NO. Income-tax Reference No. 87 of 1972 ATTORNEY(S) JUDGES Vimadalal S.K Desai, JJ. ACTS provisions of the Portuguese Civil Code read with article 10 of the Commercial Code.20. provisions of the Portuguese Civil Code and article 10 of the Commercial Code, provisions of articles 1113 to 1116 of the Portuguese Civil Code SECTION 26 INCOME TAX ACT 1961 provisions of section 9(3) of the Indian Income-tax Act, 1922, Civil Code read with article 10 of the Commercial Code, Civil Code, and article 10 of the Commercial Code section 9(3) of the Indian Income-tax Act, 1922, provisions of the Indian Partnership Act, section 48 of the Indian Partnership Act. INCOME TAX ACT 1961 section 256(1) of the Income-tax Act, Income-tax Act, and that section 41 Article 10 of the Commercial Code SECTION 41 INCOME TAX ACT Civil Code.6. Article 1096 provisions of section 9(3) Indian Partnership Act, INCOME TAX ACT Commissioner Of Income-Tax, Mysore v. Purushotam Gangadhar Bhende. Vimadalal, J.:— The assessee is a citizen of Goa, governed by the Portuguese Civil Code. He was married as per the custom of Goa, and under the relevant provisions of the Portuguese Civil Code, a house property which yielded an income of Rs. 3,810 during the assessment year 1968-69, with which the court is concerned in the present reference, thereupon became the property of the communion of the husband and wife. It will hereafter be referred to as “communion property”. The Income-tax Officer deter-mind the status as “body of individuals” of husband and wife and taxed the income from the house property accordingly. That decision was reversed by the Appellate Assistant Commissioner who held that the respective shares of the husband and wife must be taxed in their individual hands separately undersection 26 of the Income-tax Act, 1961.On appeal to the Tribunal, on a consideration of the relevant provisions of the Portuguese Civil Code, it has held that each of the consorts had a vested interest in his or her half share, and by its order dated 13th December, 1970, the Tribunal, therefore, confirmed the Appellate Assistant Commissioner's view that the income from the house property was liable to assessment in equal shares in the hands of each of the consorts separately and dismissed the appeal. On this reference, the following question has been referred to the High Court undersection 256(1) of the Income-tax Act,1961: “Whether, on the facts and in the circumstances of the case, the Tribunal was right in law in holding that the income from the house property possessed by the communion of interest of husband and wife married under the Portuguese Civil Code was liable for assessment in equal shares in the hands of the ‘body of individuals’ of communion of husband and wife for the relevant assessment year?” 2. When the matter first came up before us, parties were not agreed in regard to the proper translation of the relevant provisions of the Portuguese Civil Code. It was, therefore, adjourned for obtaining an official translation from Goa. When it came up before us again on 10th December, 1974, an official translation was produced, and was tendered and marked by us as exhibit “A”. That was supplemented by an agreed translation of certain other provisions of the Portuguese Civil Code on which also the parties sought to rely, and the same was tendered and marked exhibit “B” by us. 3. I must, at the outset, refer to the relevantprovisions of the Income-tax Act, 1961,with which we are concerned in this reference. Section 2(31) defines the term “person” as including, inter alia, an individual as well as an association of persons or a body of individuals, whether incorporated or not. Section 14 enacts that all income must be classified for the purpose of charge of income-tax and computation of total income under the six heads of income set out therein, one of which is “income from house property”. The Act then proceeds to enact appropriate provisions for the computation of the total income relating to each of the heads listed in section 14, and sections 22 to 27 deal with the head of income from house property. Section 26 which occurs in that group of sections is important for the purpose of the present case and must be set out verbatim. It reads as follows: “Where property consisting of buildings or buildings and lands appurtenant thereto is owned by two or more persons and their respective shares are definite and ascertainable, such persons shall not in respect of such property be assessed as an association of persons, but the share of each such person in the income from the property as computed in accordance with sections 22 to 25 shall be included in his total income.” 4. It may be mentioned thatsection 26 of the Income-tax Act, 1961,reproduces what wassection 9(3) of the Indian Income-tax Act, 1922,which was introduced into that Act some time in the year 1939. In connection with the language employed by the legislature in section 26, it must be noted that whilst the word “definite” used therein has a connotation in praesenti, the word “ascertainable” has a connotation in futuro, the legislature having advisedly not used the word “ascertained”. In the Oxford English Dictionary, volume III, several meanings of the word “definite” have been given, but, in my opinion, in the context in which that word is used in section 26, the meanings which are applicable are from the first meaning given in the said dictionary, and they are “fixed, certain”. When the word “definite” is understood in the sense of “fixed”, it means fixed as opposed to fluctuating, and when the word “definite” is understood in the sense of “certain”, in my opinion, it means that it is certain in the sense of being not dependent on a contingency, or in the sense of not being in dispute. The case ofAbdul Rahman v. Commissioner of Income-tax, [1944] 12 ITR 302 (Lah) was a case in which the parties were Mahomedans. The brothers disputed the right of the sisters to any share in the estate left by their deceased father by reason of the customary law alleged to be applicable to them, and there was a suit pending between them. The view taken was (at page 309) that though the share of each heir could be said to be ascertainable, so long as litigations were pending, it could not be said to be definite within the ordinary meaning of that term. The term “ascertain” is also given several meanings in volume I of the Oxford English Dictionary, but the meaning which, in my opinion, is applicable in the context in which it is used in section 26 is part of the 8th meaning given therein, viz., to make precise by determining exactly its limits, extent, amount, etc. As already pointed out above, section 26 uses the word “ascertainable” which would, therefore, mean something capable of being made precise by determining its limits, extent or amount. The question which we have to determine in this reference, therefore, is whether in the case of a husband and wife governed by the Portuguese Civil Court and married according to the custom of Goa, each of them can be said to own shares in house property which are “definite and ascertainable” within the above meaning of the terms as used in section 26. Mr. Joshi sought to contend that the shares of the husband and wife must be definite and ascertainable in respect of each individual house property, but I am not prepared to accept that contention of Mr. Joshi. First and foremost, it is important to bear in mind that section 26 deals with one of the heads of income in section 14 which is captioned as “income from house property” and which must mean the total income from all the house properties owned by the assessee. Secondly, the opening words of section 26 itself show that the section deals with property “consisting of buildings or buildings and lands appurtenant thereto owned by two or more persons”, which must mean that it deals with property in a collective sense and not with a specific property. The use of the word “property” in the latter part of the same section must, therefore, obviously be given the same meaning and must be held to have been used in a collective sense as referring to the income of all the house properties owned by the assessee. I, therefore, hold that the definite and ascertainable shares which the assessee must hold in order to come within section 26 need not be definite and ascertainable shares in each individual house property, but in all the house properties owned by him collectively, regardless of the assessee's income from the other heads of income mentioned in section 14 with which section 26 is not concerned. The second question that arises, as a matter of construction of section 26, is whether the shares of the assessee must be definite and ascertainable in the corpus of the house properties, or in the income thereof, or in both the corpus as well as the income of the house properties. A plain reading of the section, in my opinion, shows that the opening part clearly contemplates that the assessee must have definite and ascertainable shares in the corpus of the house property owned by him, and the latter part of the section, when it refers to the income from that house property, postulates that the income is also to be owned by the assessee in the same definite and ascertainable shares as the corpus. The view I take of section 26, therefore, is that in order to fall within section 26, the assessee must have definite and ascertainable shares in the corpus as well as the income of all the house properties which he owns. 5. I must next proceed to consider the relevant articles of the Portuguese Civil Code, translations of which have been tendered and marked exhibits “A” and. “B” before us, as already stated above. Article 373 lays down that “things” are either immovable or movable; article 375 then sets out what are “immovables” by virtue of law; article 376 enacts that all material things which are not immovable properties are “movable” and clause “one” of article 377 lays down that by the words “movable things or movable estates” must be understood only material objects which by their nature are movable. These articles, in my opinion, show that money is not included within the term “movables” as used in the Portuguese Civil Code. 6. Article 1096 which occurs under the heading “general provisions” permits consorts to stipulate before the celebration of marriage and within the limits prescribed by law whatever they think proper in respect of their estate, and article 1097 provides that such agreement must be by way of a public deed. Article 1098 enacts that in the absence of any such agreement, the marriage shall be deemed to have been celebrated as per the custom of the country and article 1099 lays down that in such cases the provisions of article 1108 to 1124 would be applicable. It is an agreed position between the parties that the marriage in the present case was a marriage celebrated as per the custom of the country and articles 1108 to 1124 are applicable, as far as the husband and wife in the present case are concerned. 7. Article 1108 lays down that marriage as per the custom of the country consists in communion between the consorts, of all their estates, present and future, not excluded by law; and article 1109 enlists what is excluded from the communion. It is not necessary to deal with the same, but it is pertinent to note that in the unique para, (proviso) it is laid down that the incommunicability of the estate mentioned in article 1109 does not cover the fruits of and the income from the estate. Articles 1110 to 1116 are a group of articles dealing with debts, and amongst them, article 1113 provides that debts are communicable when acquired during the subsistence of the matrimony by the act or conduct of both the consorts, or by the husband with the consent of the wife, or by the wife with the authorisation of the husband, or by the wife alone in a case falling under article 1116 with which we are not concerned. Article 1114 provides that the personal estate of the husband is liable for repayment of debts acquired by him without the consent of the wife during the subsistence of the matrimony, and that in the absence of any personal estate of the husband, the same are to be repaid “from his half share in the common estate”, though immunity from recovery is granted in such cases till the dissolution of the marriage or separation of the estate between the consorts. 8. That brings me to article 1117 which is a very important provision for the purpose of the present case. Article 1117 enacts in unequivocal terms that “the dominion and possession” of the common estate vest in both the consorts during the subsistence of the matrimony, though the management of the estate of the couple belongs to the husband. The same article, however, provides, that the wife may manage the estate of the couple by consent of the husband, or during his absence or his suffering from some impediment. The word “dominion” has its derivation from the term “dominium” which is a term of Roman law often retained in legal use and means “lordship” or “ownership”. The Shorter Oxford English Dictionary, 3rd edition, therefore, gives at page 551, the meaning of the word “dominion” in law (third meaning) as being “ownership; property; right of possession, and that is the sense in which the word” dominion “is used in article 1117. Article 1118 empowers the husband to freely dispose of the movable properties of the couple, but in case he alienates or binds under a gratuitous contract any such property without the consent of the wife, the value of the properties so alienated is to be” taken into account “towards the husband's half share. The use of the words “dispose”, “alienates” and “contracts”, as well as the definition of the term “movable things” or “movable assets” in articles 373 to 377 referred to above, in my opinion, show that article 1118 does not apply to money and cannot, therefore, apply to the income from communion property which the husband may receive in the form of cash. What is more, article 1471 shows that where the legislature of Goa intended to refer to money, it has provided for it in distinct terms and has not included it in the term “movables” as used therein. Similarly, article 1119, which relates to immovable properties, whether belonging exclusively to either of the consorts or belonging to them in common, provides that they cannot be alienated or any charge created thereon without common consent and agreement. Article's 1118 and 1119, therefore, in my opinion, also show that the wife has certainly an interest both in the movable as well as the immovable properties of the communion. Indeed, Mr. Joshi has not contended that communion properties belong to the husband only, but the whole dispute in this reference has been in regard to the question as to whether, during the subsistence of the communion, the share of either spouse can be said to be definite and ascertainable within the meaning of those terms insection 26 of the Income-tax Act, 1961. Article 1120lays down that the husband is not allowed to repudiate any estate without the consent of the wife, but the liability, if any, for “pure acceptance”, without the consent of the wife, would fall only on his half share and on the estate belonging exclusively to him. 9. Articles 1121 to 1124 are again important in so far as they deal with what happens on the termination of the communion of husband and wife. Article 1121 lays down that the communion terminates by dissolution of the marriage or by separation in conformity with law; and article 1122 enacts in the clearest possible terms that in case of death of one of the consorts, the survivor is to hold and manage the conjugal estate till the finalisation of the partition which in the language of Indian law means that the survivor would be a constructive trustee for the heirs or legatees of the deceased spouse till partition. Article 1123 then lays down that on partition between the consorts or their heirs, the property is to be divided equally and each spouse is to pay what he or she owes to the common estate and the next article 1124 provides that the wife is to be given credit for what is due to her prior to the payment of the credits of the husband, and in case the common estate is insufficient for payment of her entire claim, the husband is liable to pay the same out of his own estate, except in the case of a debt not imputable to him. This article further provides that the husband is not to enjoy a similar right of claim against the personal estate of the wife, presumably because it is he who has been in management of the communion property and is, therefore, responsible for the situation contemplated by article 1124. 10. Article 1189 states that the management of all the properties of the conjugal society belongs to the husband, but in the case of his absence or his suffering from any impediment, the wife may manage the same. Articles 1191 and 1193, however, impose restrictions on the rights of management of the husband or the wife, as the case may be; article 1191 provides that the husband cannot, in the course of his management, alienate immovable properties or move the court in respect of any dispute relating to the same, without the consent of the wife. Article 1203 lays down that the conjugal society can be interrupted either with regard to person or property of the consorts, or only with regard to property. Article 1204 states that it is permissible to the spouses to obtain the separation of persons and assets on the same grounds on which they could obtain a divorce under the law applicable to them, but article 1210 lays down that the separation of persons necessarily means a separation of estate, with the result that whilst there can be a separation of their estate simpliciter, there cannot be separation of persons without the necessary consequence of the separation of estate also. Article 1216 provides that the disposition between living consorts of the immovable properties which have devolved on each of them after separation depends on the consent of both, and in case either of them unreasonably withholds his or her consent, that difficulty can be solved by approaching the court. Article 1219 gives the wife a right to apply for a separation of estate simpliciter in a case in which she finds herself in manifest danger of losing her property due to maladministration by her husband. Article 1220 provides that the separation may fall on the estate which the wife might have brought to the conjugal society or which may have devolved to her thereafter, and on the half share of the properties which the spouses have acquired during matrimony. Article 1226 lays down that the separation of estate simpliciter does not release the wife from her obligation to contribute towards the expenses of the couple out of the income of her properties in proportion to her assets in relation to the assets of the husband. In order to understand these articles in their proper perspective, it must be remembered that they relate to a situation in which, though the spouses are separate in estate, the matrimonial bond continues. Article 1471 states that gifts of movables or money made by the husband without the consent of the wife must be placed to the account of his moiety, except in a case in which they are by way of reward, or are of little importance. The last article of the Portuguese Civil Code to which I need refer is article 1766 in which it is provided that persons married as per the custom of the country are not allowed, under the penalty of the transaction being null, to dispose of specific properties belonging to the conjugal society, except when such properties have been allotted to that spouse on partition, or the other spouse has consented to it, or in certain other cases to which it is unnecessary to refer.Article 10 of the Commercial Codeenacts that the payment of the commercial debts of the husband, which are to be made through his half share in the common assets, can be prayed for before the dissolution of the marriage or separation; and the wife must be summoned so that, if she so desires, she may apply for judicial separation of the assets within the period provided in that article. 11. In my opinion, the only articles that need consideration in support of Mr. Joshi's contention that the shares of the husband and wife in communion property are not definite and ascertainable within the terms ofsection 26 of the Income-tax Act, 1961, are articles 1104, 1216and 1220. In my opinion, however, none of the said three articles really help Mr. Joshi. Article 1104 applies only to the particular case of an ante-nuptial agreement in a marriage under articles 1096 and 1097, and can have no application to the present case which is not a case of such an agreement that is one of a marriage according to the custom of Goa. In the case of a separation of estate, article 1220 provides for the reciprocal divesting of properties brought in at the time of the marriage, but with regard to properties acquired during the marriage, it adopts the only practical course of a reciprocal vesting of half of it in each of the spouses. Article 1216 does not militate against the assessee's contention because there being only a separation, the bond of matrimony continues to subsist and the power of disposal is, therefore, on the lines of that under articles 1119, 1191 and 1193 which were applicable prior to that separation. 12. On a careful consideration of all the above provisions of the Portuguese Civil Code, as well as ofArticle 10 of the Commercial Code, the following legal propositions emerge t (i) During the subsistence of a marriage celebrated as per the custom of Goa, the ownership and possession of “the common estate”, immovable as well as movable, vests in both the husband as well as the wife. This is laid down in express terms in article 1117. Articles 1118 and 1119 as well as 1766 are also consistent with that legal position; (ii) Proposition No. 1 applies to the corpus as well as the income of all communion property, immovable as well as movable. The unique para, (proviso) to article 1109 lays down that even the income of property excluded from the communion is communion property. A fortiori the income from the communion property itself must be communion property; (iii) Under articles 1117 and 1189, the husband has only a right of management, but even that right is not an absolute right so as to amount to the “ownership” of the income, in view of the provisions of articles 1118, 1119, 1191 and 1219. Moreover, under the very articles 1???7 and 1189, even the wife can be in management in certain contingencies, her right being similarly fettered under the provisions of article 1193; (iv) In the corpus as well as the income of communion property, immovable as well as movable, the husband and the wife each have, during the subsistence of a marriage celebrated as per the custom of Goa, a fixed and certain half share which can be ascertained on the termination of the communion by divorce, separation or death (articles 1121 to 1124, 1203, 1204, 1210, 1216, 1220 and 1226). What is most important in this connection is that it is an admitted position that on the death of one of the spouses, communion property does not devolve by survivorship, but the half share of the deceased spouse goes by succession to his or her own heirs or legatees by virtue of articles 1122 and 1123. There is a consistent reference to the half share of each of the consorts throughout the different articles dealing with various situations (vide articles 1112 to 1114 of the PortugueseCivil Code, and Article 10 of the Commercial Codedealing with the incidence of debts, and Portuguese Civil Code article 1118, dealing with the disposal of the movable property as well as articles 1120, 1123, 1220, 1463, and 1471). 13. Mr. Joshi on behalf of the Commissioner formulated two propositions in support of his case, and they are as follows: (1) In communion properties, or their income, the husband and wife have each no definite and ascertainable share so long as the communion lasts; and (2) If he is wrong in that view, in any event, the husband and wife do not each have a definite and ascertainable share in each individual property. 14. In support of these propositions, Mr. Joshi contended that under the Portuguese Civil Code the position of those married according to custom was akin to that of coparceners in a Hindu joint family, with only this difference that there is no right of survivorship as in the latter case. In that connection, Mr. Joshi relied upon the decision of the Madras High Court in the case ofArunachala Mudaliar v. Commissioner of Income-tax, [1961] 41 ITR 432 (Mad) in which there was a severance of status by a preliminary decree in a suit for partition of a Hindu joint family. Two of the sons were appointed joint receivers and were directed to divide the income among the father and sons equally, and to pay certain allowances. An application was made to the Income-tax Officer under section 25A(1) of the Act claiming that by reason of the preliminary decree a partition had taken place, but that application was rejected. The receivers were assessed undersection 41 of the Income-tax Actas representing the Hindu undivided family on the ground that there was no division by metes and bounds. The Tribunal held that there being no order under section 25a(1), the hindu undivided family must be deemed to exist for the purpose of theIncome-tax Act, and that section 41was not applicable to the case. On a reference to the High Court, what was held (at pages 444-445) was that theIncome-tax Actclearly distinguished between an association of persons and a Hindu undivided family, thatsection 9(3) of the Indian Income-tax Act, 1922,did not apply as it could not override the special provision relating to a Hindu undivided family contained in section 25A, and that the Hindu undivided family was the owner for the purpose of assessment to tax so long as there was no partition in definite portions even when there had been a severance in status. I am afraid that the decision of the Madras High Court cannot be of any assistance to Mr. Joshi in the present case, for the simple reason that all that was held was that in the case of a Hindu undivided family section 9(3) could not be applied in view of the special provision in section 25a relating to partition. Since there had been no partition by metes and bounds, it was in view of section 25A that the claim to the applicability of section 9(3) was negatived by the Madras High Court. The special situation that prevailed in the said case can, therefore, bear no analogy to the present case. Mr. Joshi next contended that the position of the communion of the husband and wife in the present case is akin to that of partners during the subsistence of a firm under theIndian Partnership Act,and in support of that contention, he relied on the decision of the Rajasthan High Court in the case ofNew Cotton & Wool Pressing Factory v. Commissioner of Income-tax, [1967] 65 ITR 662 (Raj) in which immovable properties were owned by a firm, and it was held thatsection 9(3) of the Indian Income-tax Act, 1922,had no application to such a case. In arriving at that conclusion, the Rajasthan High Court correctly held (at pages 667-669), on a review of the provisions of the Partnership Act, and particularly section 48 thereof, that a partner cannot claim any definite share in the property of the firm so long as the firm was not dissolved and accounts settled as provided in section 48 of that act. That view of the nature of a partner's interest during the subsistence of a firm, whatever be the character of the property brought in, is now confirmed by the Supreme Court in the case ofAddanki Narayanappa v. Bhaskara Krishnappa,AIR 1966 SC 1300, para. 3, in which, after referring to the relevantprovisions of the Indian Partnership Act,the Supreme Court held that during the subsistence of a partnership no partner can deal with any portion of the property as his own, and that his right is merely a right to obtain such profits, if any, as fall to his share from time to time until dissolution, and, thereafter, to a share in the assets of the firm remaining after satisfying its liabilities as set out insection 48 of the Indian Partnership Act.That view was reaffirmed by the Supreme Court itself in the later case ofCommissioner Of Income Tax, West Bengal, Calcutta v. Juggilal Kamalapat, [1967] 63 ITR 292 (SC). The legal position that entails in the case of partners during the subsistence of a firm has, therefore, no application to a case like the present one in which it could never be said that the husband or wife had no definite share of the immovable property of the communion during its subsistence. The reason why the Rajasthan High Court refused to apply theprovisions of section 9(3) of the Indian Income-tax Act, 1922,is, therefore, wholly inapplicable in the case of the communion of husband and wife under the Portuguese Civil Code. 15. Mr. Joshi next contended that the expression “definite and ascertainable” in section 26 contemplated a right in praesenti to partition and separate enjoyment, and in support of that proposition, he relied on the decision of the Supreme Court in the case ofCommissioner of Income-tax v. Indira Balkrishna, [1960] 39 ITR 546 (SC). The facts of that case were that one Balkrishna Purani died leaving behind him three widows and three daughters, and the three widows as legal heirs inherited the estate of the deceased which consisted of immovable properties, shares in joint stock companies, money lying in deposit, and share in a registered firm. The Income-tax Officer took the status of the assessee as an association of persons and assessed them accordingly. On appeal to the Appellate Assistant Commissioner, it was held that, in any event, the income from property ought to have been assessed separately in the hands of the three widows undersection 9(3) of the Indian Income-tax Act, 1922.On further appeal to the Tribunal, it was held that the Appellate Assistant Commissioner was wrong in applying section 9(3), as the three widows inherited and possessed the property of the deceased as joint tenants, and its income was, therefore, liable to be assessed in their hands in the status of an association of persons. On a reference to the High Court, the view taken by the Tribunal was reversed, and it was held that the three widows could not be assessed in the status of an association of persons with regard to the income which they earned as the heirs of their deceased husband. On appeal to the Supreme Court, that decision of the High Court was confirmed. It was observed by the Supreme Court that the position of co-widows under Mitakshara law was well-settled; that they succeeded as co-heirs to the estate of their deceased husband and took as joint tenants with rights of survivorship and equal beneficial enjoyment; and that they were entitled as between themselves to an equal share of the income. It was further held (at page 549) that though they took as joint tenants, no one of them had a right to enforce “absolute partition” of the estate against others so as to destroy their rights of survivorship, but they had the right to obtain a partition of separate portions of the property so that each might enjoy her equal share of the income separately, though they had not chosen to do so in the said case. The Supreme Court pointed out (at page 550) that the Appellate Assistant Commissioner had held that section 9(3) applied to the income from the immovable properties in the said case and the department had not appealed from that decision to the Tribunal, and it was, therefore, not open to the Tribunal to go behind it. The Supreme Court, however, went on to state that the Tribunal was wrong in thinking that the respective shares of the widows were not definite and ascertainable and that, in its opinion, each of the co-widows had an equal share in the income, viz., ⅓rd, and theprovisions of section 9(3)clearly applied in respect of the immovable properties. We are bound by this clear expression of opinion by the Supreme Court. In my opinion, the decision of the Supreme Court in Indira's case, [1960] 39 ITR 546 (SC), far from helping Mr. Joshi, provides a complete answer to the contention of the revenue in this reference, because in that case, notwithstanding the fact that the co-widows' interest in their deceased husband's estate was not fixed absolutely but could fluctuate within certain limits, and notwithstanding the fact that there was under M???takshara law a right of survivorship among the co-widows, they were held to have “definite and ascertainable” shares in the estate of the deceased. A fortiori, the shares of the husband and the wife in the present case who, under the Portuguese Civil Code, have no right of survivorship in the communion property and whose shares therein cannot fluctuate in any contingency, must be held to be “definite and ascertainable”. Though in Indira's case, [1960] 39 ITR 546 (SC), the co-widows had a right in praesenti to partition, the decision of the Supreme Court was not based on that ground. On the contrary, it lays down that the right to partition need not be absolute. The decision of the Supreme Court in the said case, according to me, establishes the following proposition:— (1) The court is not concerned with the label “joint tenants” or “tenants-in-common”, or with the fact that possession and management may be joint, or with the fact of the absence of a right to “absolute partition”; (2) The fact that the co-widows had an equal share in the income of all the immovable properties was sufficient to attract the applicability of section 9(3). This shows that Mr. Joshi's contention that in order to attract the applicability of section 26 (which corresponds to the old section 9(3)), the share of each must be definite and ascertainable in respect of each individual property, cannot be accepted; (3) The share in the corpus also must be definite and ascertainable though it need not be absolute. It is sufficient even if the right to partition of that definite and ascertainable share is restricted, or is available for a limited purpose only, as, for instance, for the purpose of enjoying the income separately, as was the position in Indira's case, [1960] 39 ITR 546 (SC). 16. In Salmond on Jurisprudence, 12th edition, pages 254-255, it is stated that co-ownership may assume different forms, and that the two chief kinds of co-ownership in English law were “in common” and “joint ownership”. It is pointed out there that the most important difference between these two relates to the effect of the death of one of the co-owners. In ownership-in-common the right of a dead man descends to his successors like any other inheritable right; but on the death of one of two joint owners his ownership dies with him, and the survivor becomes the sole owner by virtue of his right of survivorship. Though it is not necessary to give a definite label to the relationship of the husband and wife during the subsistence of the communion, it is important to bear in mind that there is no right of survivorship between them and that their relationship under the Portuguese Civil Code is, therefore, more akin to a tenancy-in-common than to joint tenancy as known in English law. In Cheshire's Modern Law of Real Property, 11th edition, pages 327 to 337, it is stated that at common law there were four possible forms of co-ownership, one of which is now defunct, and the remaining three are joint tenancy, tenancy-in-common and coparcenary, of which the last one now seldom arises in England. Cheshire also states that the right of survivorship is one of the two essential attributes of joint tenancy as distinguished from tenancy-in-common. It is further stated there that in a tenancy-in-common each has a share in the ordinary meaning of that word, and that share is undivided in the sense that its boundary is not yet demarcated, but nevertheless his right to a definite share exists. Cheshire then points out that in the tail dying intestate leaving only female heirs, those female heirs succeeded jointly to the estate as a single heir, and were called coparceners. Like joint tenants, the coparceners had a unity of title, interest and possession, but like tenants-in-common their estate was unaffected by the doctrine of survivorship. There appears to me to be some resemblance between this concept of the English coparcenary and the position of the husband and wife during the subsistence of the communion under the Portuguese Civil Code. 17. Mr. S.P Mehta on behalf of the assessee relied on the decision of the Calcutta High Court in the case ofCommissioner of Income-tax v. Smt. Bani Rani Rudra, [1966] 59 ITR 216 (Cal), which was a case of co-heirs under the Dayabhaga law in regard to whomsection 9(3) of the Indian Income-tax Act, 1922,was applied even whilst the family was undivided. The facts of that case were that on the death of one J.K Rudra, his son and wife succeeded to the estate in equal shares. The question before the income-tax authorities was whether the income from the house properties could be assessed in the hands of the family or whether it should be treated as income in the hands of the individual members constituting the family. After referring to the legal position under Dayabhaga law under which the sons do not acquire any interest by birth and each coparcener has, even whilst the family remains undivided, a certain definite share in the joint property of which he is the absolute owner, the Calcutta High Court held that since on the death of J.K Rudra, his widow became entitled to a???oiety of the property with his son, section 9(3) was applicable and the income could not be assessed in the hands of the assessee-Hindu undivided family even if there was no partition between the widow and her son. It must be noted that under Dayabhaga law which was applicable to the parties in the said case, the coparcenary was first formed on the death of the said J.K Rudra, and as between themselves the widow and the son of the deceased held his property as coparceners, each of them having the absolute power to dispose of his or her share. There is, therefore, also some similarity between the position of co-heirs under Dayabhaga law and the position of the husband and wife during the subsistence of the communion under the Portuguese Civil Code, with this difference that there is no power of disposal in each of them in the latter case as in the former. 18. The result of a consideration of these authorities is that none of them deal with a legal position which is exactly the same as that of a husband and wife during the subsistence of the communion under the Portuguese Civil Code, and apart from the indirect guidance that one can get from the judgment of the Supreme Court in Indira's case, [1960] 39 ITR 546 (SC) cited above, none of them are of any direct assistance for the purpose of answering the question posed in this reference. 19. Mr. Joshi contended that if the income of the property held in communion is not assesssd as the income of the “body of individuals” of the husband and wife, but is assessed separately in the hands of each spouse, it will be a personal debt of each spouse, and having regard to theprovisions of articles 1113 to 1116 of the Portuguese Civil Codethe department will not be entitled to recover it from the communion property, except on partition. This would ordinarily be a good argument on the principle of harmonious construction between two statutes, or on the ground that the legislature could not have intended such an inconvenient result to follow. Those principles of construction have, however, no application to the construction of the provisions of two different systems of jurisprudence enacted by the legislatures of two different countries which, in the unusual situation that has arisen by reason of the merger of Goa in India, may well be irreconcilable. I, therefore, hold that the difficulty in effecting the recovery of tax is no ground for placing the construction urged by Mr. Joshi, if the contrary position is clear on a proper construction of the relevant provisions of the Portuguese Civil Code read withArticle 10 of the Commercial Code. 20. In the result, I hold that under the PortugueseCivil Code read with Article 10 of the Commercial Code,in the case of a husband and wife married according to the custom of Goa, each of them has a definite and ascertainable share in the corpus as well as in the income, the management alone being with the husband. As Mr. Mehta has rightly contended, restrictions on the enjoyment or management of the property which are to be found in the Portuguese Civil Code do not contravene any requirement ofsection 26 of the Income-tax Act, 1961.The Tribunal was, therefore, right in the view which it took that section 26 applied, and the question referred to us must be answered against the Commissioner. S.K Desai, J.:— I agree and have nothing to add. By the Court.— We answer the question referred to this court as follows: On the facts of the case, and having regard to the relevantprovisions of the Portuguese Civil Code and Article 10 of the Commercial Code,the respective half shares of the husband and wife in the income from the house property which is the property of the communion of the husband and wife married according to the custom of Goa, should be assessed separately in equal shares in the hands of each of them, and not in the hands of “the body of individuals” of the communion of husband and wife, for the relevant assessment year. 21. The Commissioner must pay the costs of the reference to the assessee.	https://www.casemine.com/judgement/in/5608f980e4b01497111450e5
Mean International Wealth Index (IWI) score of region - Area Database - Region Ranking - Global Data Lab Area Database (v4.2.1) Mean International Wealth Index (IWI) score of region First decile Country / Region Angola / Bie Angola / Cunene Angola / Kuanza Sul Angola / Uige Burkina Faso / Boucle de Mouhoun Burkina Faso / Est Burkina Faso / Sahel Burkina Faso / Sud-Ouest Burundi / East (Cankuzo, Rutana, Ruyigi ) Burundi / Middle (Gitega, Karuzi, Muramvya, Mwaro) Burundi / North (Kayanza, Kirundo, Muyinga, Ngozi) Central African Republic CAR / RS I (Ombella Mpoko, Lobaye, Kemo, Nana Grebizi, Ouaka) Central African Republic CAR / RS II (Mambera Kadei, Nana Mambere, Sangha Mbaere) Central African Republic CAR / RS III (Ouham Pende, Ouham) Central African Republic CAR / RS IV (Haute-Kotto, Baminigui Bangoran, Vakaga) Central African Republic CAR / RS V (Basse Kotto, Mbornou, Houte Mbormou) Chad / Zone 2 (Borkou, Ennedi, Tibesti, Kanem, Barh El Gazal, Lac) Chad / Zone 3 (Guera, Batha, Salamat) Chad / Zone 4 (Ouaddai, Assongha, Sila, Biltine - Wadi Fira) Chad / Zone 5 (Chari-Baguimi, Dababa, Baguirmi, Hadjer Lamis) Chad / Zone 6 (Mayo-Kebbi Est and Ouest) Chad / Zone 7 (Logone Occidental & Oriental, Monts de Lam, Tandjile Est & Ouest) Chad / Zone 8 (Mandoul, Moyen-Chari, Bahr Koh, Lac Iro) Congo Brazzaville / Cuvette Ouest Congo Brazzaville / Kouilou Congo Brazzaville / Lekoumou Congo Brazzaville / Likouala Congo Brazzaville / Pool Congo Democratic Republic / Bandundu Congo Democratic Republic / Equateur Congo Democratic Republic / Kasai Occidental Congo Democratic Republic / Kasai Oriental Congo Democratic Republic / Maniema Congo Democratic Republic / Orientale Equatorial Guinea / Centro Sur Equatorial Guinea / Kie Ntem Equatorial Guinea / Wele Nzas Eritrea / Anseba Eritrea / Debub Eritrea / Gash-Barka Eritrea / Semenawi Keih Bahri Ethiopia / Affar Ethiopia / Amhara Ethiopia / Ben-Gumz Ethiopia / Oromiya Ethiopia / SNNP Ethiopia / Somali Ethiopia / Tigray Guinea 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Lindi Tanzania / Mtwara Tanzania / Rukwa, Katavi Tanzania / Singida Uganda / Central (Central 1, Central 2, Kampala) Uganda / Eastern (Eastern, East Central) Uganda / Northern (North, Karamoja, West Nile) Uganda / Western (Western, Southwest) Uganda / Eastern (Bukedi, Bugishu, Teso) Uganda / North (Karamoja, Lango, Acholi) Uganda / West Nile Vanuatu / Torba Zambia / Northern Zambia / Western Second decile Country / Region Angola / Huambo Angola / Huila Angola / Kuando Kubango Angola / Kuanza Norte Angola / Lunda Norte Angola / Moxico Benin / Borgou (incl Alibori) Benin / Mono (incl Couffo) Burkina Faso / Centre-Est Burkina Faso / Centre-Nord Burkina Faso / Centre-Ouest Burkina Faso / Centre-Sud Burkina Faso / Nord Burkina Faso / Plateau Central Burundi / South (Bururi, Makamba) Burundi / West (Bubanza, Buja Rural, Cibitoke, Mairie de Bujumbura) Cameroon / Extreme Nord Cameroon / Nord Congo Brazzaville / Bouenza Congo Brazzaville / Plateaux Congo Brazzaville / Sangha Congo Democratic Republic / Bas-Congo Congo Democratic Republic / Katanga Congo Democratic Republic / Nord-Kivu Congo Democratic Republic / Sud-Kivu Ethiopia / Gambela Guinea / Faranah Guinea / Labe Guinea / N Zerekore Guinea Bissau / Biombo Guinea Bissau / Cacheu Guinea Bissau / Gabu Guinea Bissau / Oio Guinea Bissau / Quinara Guinea Bissau / Tombali Haiti / Artibonite Haiti / Camps Haiti / Centre Haiti / Grande-Anse, Nippes Haiti / North-West Haiti / South Haiti / South-East Indonesia / East Timor Kenya / Nyanza Kenya / Western Kiribati / Central Gibert Kiribati / Northern Gilbert Kiribati / Southern Gilbert Lesotho / Mokhotlong Country / Region Lesotho / Thaba-Tseka Liberia / South Central Liberia / Bomi Liberia / Grand Bassa Liberia / Grand Gedeh Liberia / Lofa Liberia / Margibi Liberia / Maryland Liberia / Nimba Liberia / River Gee Liberia / Sinoe Madagascar / Mahajanga Madagascar / Alaotra Mangoro Madagascar / Atsinanana Madagascar / Betsiboka Madagascar / Boeny Madagascar / Bongolava Madagascar 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Papua New Guinea / Western Papua New Guinea / Western Highlands Rwanda / East Rwanda / North Rwanda / South Rwanda / West Sierra Leone / Bo Sierra Leone / Bombali Sierra Leone / Kambia Sierra Leone / Kenema Sierra Leone / Kono Sierra Leone / Port Loko Somalia / Awdal Somalia / Sool Somalia / Togdhere Sudan / Al Gedarif Sudan / Kassala Tanzania / Arusha, Manyara Tanzania / Iringa, Njombe Tanzania / Mara Tanzania / Mbeya Tanzania / Morogoro Tanzania / Mwanza, Geita Tanzania / Pwani Tanzania / Ruvuma Tanzania / Shinyanga, Simiyu Tanzania / Tabora Togo / Savanes Uganda / East Central (Busoga) Uganda / Southwest (Ankole, Kigezi) Uganda / Western (Bunyoro, Tooro) Vanuatu / Malampa Vanuatu / Penama Vanuatu / Sanma Vanuatu / Tafea Zambia / Eastern Zambia / Luapula Zambia / North-Western Zimbabwe / Matebeleland North Third decile Country / Region Afghanistan / Central Highlands (Bamyan Daikundi) Afghanistan / East (Nangarhar Kunar Laghman Nooristan) Angola / Bengo Angola / Benguela Angola / 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Equatorial Guinea / Litoral Eritrea / Debubawi Keih Bahri Ethiopia / Dire Dawa Gabon / Ogooue Ivindo Gambia / Janjabureh Gambia / Kuntaur Ghana / Upper East Ghana / Upper West Country / Region Guinea / Boke Guinea / Kankan Guinea / Kindia Guinea / Mamou Guinea Bissau / Bafata Haiti / North Haiti / North-East Honduras / Gracias a Dios Kenya / Coast Kenya / Eastern Kenya / Rift Valley Kiribati / Line and Phoenix Group Lesotho / Butha-Buthe Lesotho / Mafeteng Lesotho / Mohale s Hoek Lesotho / Qasha s Nek Lesotho / Quthing Madagascar / Antananarivo Madagascar / Antsiranana Madagascar / Toamasina Madagascar / Analanjirofo Madagascar / Diana Madagascar / Sava Malawi / Blantyre Malawi / Mzimba Mali / Kayes Mali / Koulikoro Mali / Segou Mali / Sikasso Mauritania / Assaba Mauritania / Brakna Mauritania / Hodh Gharbi Mauritania / Tagant Mozambique / Maputo Provincia Myanmar / Ayeyarwaddy Myanmar / Rakhine Namibia / Caprivi Namibia / Kunene Namibia / Omusati Namibia / Oshikoto Nepal / Karnali 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Evaluation of MR Imaging Compatibility of a New Intracranial Pressure Monitoring Device \| American Journal of Neuroradiology Research Article BRAIN Evaluation of MR Imaging Compatibility of a New Intracranial Pressure Monitoring Device Jens O. Heidenreich , Rüdiger Stendel , Mario Brock , Karl-Jürgen Wolf and Andreas M. Schilling American Journal of Neuroradiology February 2005, 26 (2) 360-362; Article Abstract Summary: We report our observations on MR imaging quality and functionality of a recently introduced intracranial pressure monitoring device. The device was tested at two different field strengths in a pig brain specimen to investigate MR imaging artifacts, probe function during and after MR data acquisition, and device-related temperature changes in the brain tissue. Image reading was not impaired, and probe function, although reduced, was not fully interrupted during data acquisition. Critically ill neurosurgical patients benefit from continuous intracranial pressure (ICP) monitoring, especially after severe head trauma, subarachnoid hemorrhage, intracerebral hemorrhage, or nontraumatic brain edema (1,2). MR imaging offers advantages over cranial CT with regard to treatment planning and outcome prediction. It differentiates cerebral structures more reliably, it distinguishes between infarction and edema, and it depicts brain contusions in earlier stages (3–7). Furthermore, brainstem lesions—which may be important in determining the prognosis and outcome—are best detected with MR imaging (8). The monitoring of such critically ill patients is crucial, and none of the commercially available ICP monitoring devices fully meets the definitions of MR safety and compatibility by the US Food and Drug Administration. The aim of this study was to evaluate the recently introduced Neurovent-P (Rehau AG+CO; Rehau, Germany) ICP monitoring device for use in MR imaging, particularly in terms of image quality, probe function and temperature induction. Observations The Neurovent-P ICP device (Rehau AG+CO) (Fig 1), described in detail elsewhere (9), was placed in a straight position with the tip in the white matter of a fresh pig brain specimen at a depth of 2 cm. The specimen was kept in a plastic box filled with physiologic NaCl solution to ensure sufficiently high signal intensity from the probe. Temperature measurements were performed at three time points immediately before, during, and after the acquisition of different MR images. For this we used two clinical MR machines with 1.5- and 0.2-T field strengths and a circularly polarized send-receive head coil (Magnetom Vision and Magnetom OPEN; Siemens, Erlagen, Germany). Two microthermistors (Rehau AG+CO) were positioned—one next to the tip and the other 5 cm away from the tip in the brain parenchyma—to monitor temperature gradients. Tip of the Neurovent-P ICP monitoring device (Rehau AG+CO) in comparison with a match. F ig2. Sagittal T1- and T2- weighted images allow good tissue discrimination despite artifact related to the ICP monitoring device. MR imaging measurements were carried out in a manner suited to neurosurgical demands, and involved sequences for assessing intracerebral or subarachnoid hemorrhage, brain edema, contusion, and infarction. Accordingly, we performed the following sequences in three planes to evaluate the image quality: T2-weighted turbo spin-echo (SE) (TR/TE = 5300/128; turbo factor 23), T2-weighted gradient-echo (GRE; fast low-angle shot [FLASH] 2D, a TR/TE of 344/52, and a flip angle of 15°), and T1-weighted SE (420/12, three acquisitions). In addition, echo-planar sequences (4295/110) were applied for their high-gradient switching rates and radio-frequency deposition in view of temperature changes. Because of the different field strengths and software releases, the sequences varied for each machine. Measurements were performed in the same manner to determine the in-magnet functionality of the ICP monitoring device. Functionality was continuously tested during MR imaging data acquisition, while we planned the next examination, and at the end of the examination. The device was thus connected to a patient monitor (Dash 3000, Marquette; Solingen, Germany) via a zero simulator NPS-2 (Rehau AG+CO). The probe was stabilized at a constantly recorded pressure of 2.4 mm Hg. Pressure measurements were recorded at a rate of 10 per second by using a digital data logger (Rehau AG+CO). Data postprocessing was performed by using commercially available software (Excel 2002; Microsoft, Redmond, WA). Although known for their high-gradient switching rates and artifact induction, transverse T2-weighted TSE images at 1.5 T showed low signal-intensity loss, with a maximum artifact diameter of 6 mm in relation to a 4-mm probe size. Tissue discrimination next to this artifact was not impaired. Similarly oriented T1-weighted images showed excellent tissue discrimination (Fig 2) despite an artifact with a maximum diameter of 15 mm. GRE sequences resolved artifacts larger than those on T2-weighted turbo SE images, with average diameters of 12, 15, and 20 mm in the axial, coronal, and sagittal planes, respectively. Imaging quality was acceptable in the axial plane, satisfactory in the coronal plane, and unsatisfactory in the sagittal plane. The axial echo-planar sequence, which is usually applied for functional imaging at our institution and which is not routinely performed in the acute phase, caused huge artifacts; the resultant image quality was unacceptable. The T2-weighted images at 0.2 T had good imaging quality regardless of the section orientation. No artifact induction was measurable with 3- and 4-mm artifacts in comparison to the tip size of 4 mm. T1-weighted SE and GRE sequences in any plane showed strong artifact induction, measuring 10 mm on T1-weighted images and 8 mm on GRE images. However, image quality was good. Temperature measurements taken from the tip of the ICP probe resolved a maximum temperature shift of −0.1°C from a starting point for the 0.2-T scanner and −0.13°C for the 1.5-T system. No relevant temperature gradients were detectable with either system. We saw no signs of movement or torque when the tip of the probe was exposed to the magnet bore of the 1.5-T system. When the entire probe was exposed to the magnet bore, the plug of the probe was pulled discretely toward the magnet forming an angle of less than 30° relative to its base. The displacement was induced by a force of 6 pounds, less than the total weight of the probe of 12.4 pounds. Gravitation thus has a stronger effect on the fixated probe than the magnetic field. MR imaging data acquisition affected the measured pressure values to various extents. No changes were detected in pressure values measured inside or outside the machine. The actual pressure was underestimated during data acquisition: The highest values roughly corresponded to the initially measured pressure. Pressure immediately returned to the originally measured value in the intervals between image acquisitions. Discussion MR imaging is routinely performed to examine neurosurgical patients. It offers the advantages of soft tissue discrimination, adding information about damage to the blood-brain barrier, early edema formation, and even metabolic (MR spectroscopic) and functional MR imaging analysis. Still, these advantages over CT often cannot be exploited in patients with severe head injury or intracranial hemorrhage. Numerous reports have described the benefit patients gain from continuous monitoring of ICP (1,2,10), and the cumulative risk of transporting severely ill patients to the MR imaging unit and disconnecting them from continuous monitoring or removing the pressure measuring device is not justified. Therefore, the demand for MR imaging-compatible continuous ICP monitoring devices is unquestioned. Various attempts have been made to develop MR imaging-safe monitoring and life-support devices for use in anesthesiology as well as MR imaging-safe and artifact-reduced tools for interventional radiology (10–12). For example, the Codman microtransducer (Codman and Shurtleff Inc., Raynham, MA) (to which our group has no access) has been shown to be MR imaging-safe at magnetic fields up to 0.5 T (1). However, this is no longer an environment satisfactory for advanced neuroradiologic imaging, as described before. Our results show that the newly developed ICP monitoring device, whose clinical safety and reliability were demonstrated in a previous publication (9), is functional in magnetic fields of up to 1.5 T in that it accurately records pressure within the machine and in the intervals between data acquisitions. It continued to do so after data acquisition. The probe itself showed no signs of movement up to 1.5 T, as has been described for the Camino fiberoptic device (Camino Neurocare; San Diego, CA) at 2.0 T (1). Also, no relevant temperature changes exceeding 0.15°C in either direction were detected during data acquisition—either distant from or next to the tip of the device. The possibility of temperature induction applies not only to ferromagnetic materials but also to any other iron material that could act as an antenna. The main interest was, of course, the imaging quality. If the imaging quality is too poor to deliver additional or more reliable information in patients in neurointensive care, the insufficiencies related to transport and monitoring cannot be justified. We found adequate imaging quality with both machines when we applied reasonably long and demand-oriented sequences. Imaging results were unacceptable with sequences applied for functional MR imaging; this finding is of interest but of little importance in intensive care patients. T2- and diffusion-weighted images for visualizing intracranial hemorrhage and ischemia, respectively, yielded acceptable results, at least in one orientation. T1- and T2-weighted images showed excellent tissue discrimination and minimum artifact induction with the device tip in the pig brain specimen. Conclusion The good-to-excellent T1- and T2-weighted imaging results obtained in a pig brain specimen definitely encourages us to perform MR imaging without the risk of adverse events. Critically ill neurosurgical patients will benefit from the additional information gained for planning therapy and predicting outcomes. For technical reasons, we have not yet been able to evaluate this ICP device in vivo, and the gap in safety examination prevents us from commenting on its potential in conjunction with neuroexcitation. References ↵ Williams EJ, Bunch CS, Carpenter TA, et al. Magnetic resonance imaging compatibility testing of intracranial pressure probes. J Neurosurg 1999 ; 91 : 706 –709 Kent DL. A blinded clinical comparison of MR imaging and CT in neuroradiology. Invest Radiol 1988 ; 23 : 327 –330 Frayne R, Goodyear BG, Dickhoff P, et al. Magnetic resonance imaging at 3.0 tesla: challenges and advantages in clinical neurological imaging. Invest Radiol 2003 ; 38 : 385 –402 Young RJ, Destian S. Imaging of traumatic intracranial hemorrhage. Neuroimaging Clin N Am 2002 ; 12 : 189 –204 Sundgren PC, Reinstrup P, Romner B, et al. Value of conventional, and diffusion- and perfusion-weighted MRI in the management of patients with unclear cerebral pathology, admitted to the intensive care unit. Neuroradiology 2002 ; 44 : 674 –680 Macmillan CSA, Wild JM, Wardlaw JM, et al. Traumatic brain injury and subarachnoid hemorrhage: in vivo occult pathology demonstrated by magnetic resonance spectroscopy may not be “ischaemic”: a primary study and review of the literature. Acta Neurochir 2002 ; 144 : 853 –862 Kent DL, Haynor DR, Longstreth WT Jr, et al. The clinical efficacy of magnetic resonance imaging in neuroimaging. Ann Intern Med 1994 ; 120 : 856 –871 Stendel R, Heidenreich J, Schilling A, et al. Clinical evaluation of a new intracranial pressure monitoring device. Acta Neurochir 2003 ; 145 : 185 –193 Steiner LA, Czosnyka M, Piechnik SK, et al. Continuous monitoring of cerebrovascular pressure reactivity allows determination of optimal cerebral perfusion pressure in patients with traumatic brain injury. Crit Care Med 2002 ; 30 : 733 –738 Graf H, Klemm T, Lauer UA, et al. Systematics of imaging artifacts in MRT caused by metallic vascular implants (stents). Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 2003 ; 175 : 1711 –1719 Received February 4, 2004. Accepted after revision May 20, 2004. Jens O. Heidenreich , Rüdiger Stendel , Mario Brock , Karl-Jürgen Wolf , Andreas M. Schilling	https://www.ajnr.org/content/26/2/360.full
Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergic Synapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells of Postnatal Hippocampus \| Journal of Neuroscience Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergic Synapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells of Postnatal Hippocampus Kyu-Hee Lee , Hyunsu Lee , Che Ho Yang , Jeong-Soon Ko , Chang-Hwan Park , Ran-Sook Woo , Joo Yeon Kim , Woong Sun , Joung-Hun Kim , Won-Kyung Ho and Suk-Ho Lee Journal of Neuroscience 16 December 2015, 35 (50) 16479-16493; DOI: https://doi.org/10.1523/JNEUROSCI.1585-15.2015 Kyu-Hee Lee 1 Cell Physiology Laboratory Department of Physiology, bioMembrane Plasticity Research Center, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea, Hyunsu Lee 1 Cell Physiology Laboratory Department of Physiology, bioMembrane Plasticity Research Center, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea, Che Ho Yang 1 Cell Physiology Laboratory Department of Physiology, bioMembrane Plasticity Research Center, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea, Jeong-Soon Ko 1 Cell Physiology Laboratory Department of Physiology, bioMembrane Plasticity Research Center, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea, Article Abstract Expression of neuregulin-2 (NRG2) is intense in a few regions of the adult brain where neurogenesis persists; however, little is understood about its role in developments of newborn neurons. To study the role of NRG2 in synaptogenesis at different developmental stages, newborn granule cells in rat hippocampal slice cultures were labeled with retrovirus encoding tetracycline-inducible microRNA targeting NRG2 and treated with doxycycline (Dox) at the fourth or seventh postinfection day (dpi). The developmental increase of GABAergic postsynaptic currents (GPSCs) was suppressed by the early Dox treatment (4 dpi), but not by late treatment (7 dpi). The late Dox treatment was used to study the effect of NRG2 depletion specific to excitatory synaptogenesis. The Dox effect on EPSCs emerged 4 d after the impairment in dendritic outgrowth became evident (10 dpi). Notably, Dox treatment abolished the developmental increases of AMPA-receptor mediated EPSCs and the AMPA/NMDA ratio, indicating impaired maturation of glutamatergic synapses. In contrast to GPSCs, Dox effects on EPSCs and dendritic growth were independent of ErbB4 and rescued by concurrent overexpression of NRG2 intracellular domain. These results suggest that forward signaling of NRG2 mediates GABAergic synaptogenesis and its reverse signaling contributes to dendritic outgrowth and maturation of glutamatergic synapses. SIGNIFICANCE STATEMENTThe hippocampal dentate gyrus is one of special brain regions where neurogenesis persists throughout adulthood. Synaptogenesis is a critical step for newborn neurons to be integrated into preexisting neural network. Because neuregulin-2 (NRG2), a growth factor, is intensely expressed in these regions, we investigated whether it plays a role in synaptogenesis and dendritic growth. We found that NRG2 has dual roles in the development of newborn neurons. For GABAergic synaptogenesis, the extracellular domain of NRG2 acts as a ligand for a receptor on GABAergic neurons. In contrast, its intracellular domain was essential for dendritic outgrowth and glutamatergic synapse maturation. These results imply that NRG2 may play a critical role in network integration of newborn neurons. dentate gyrus ErbB4 neuregulin neurogenesis synaptogenesis Introduction Neuregulins belong to a family of peptide hormones that contains epidermal growth factor (EGF)-like domain. Neuregulin-2 (NRG2) has been identified as a second member of the neuregulins with a domain structure similar to neuregulin-1 (NRG1) (Busfield et al., 1997;Carraway et al., 1997;Chang et al., 1997). Although NRG1 and NRG2 are strongly expressed early in the developing brain, especially in germinal layers (Corfas et al., 1995;Longart et al., 2004), they are expressed in distinct cell populations in the brain and display distinct subcellular distribution. In the adult brain, the expression of NRG1 becomes low and sporadic but widespread over the brain (Longart et al., 2004;Liu et al., 2011). In contrast, intense expression of NRG2 persists in three regions of an adult brain: the cerebellum, olfactory bulb, and the hippocampal dentate gyrus (Carraway et al., 1997;Longart et al., 2004). NRG2 is preferentially expressed in dendrites, whereas NRG1 is targeted to the axon as well as dendrites (Longart et al., 2004). Such distinct temporal and spatial expression patterns of NRG2 imply that it may have nonredundant biological functions. Developmental consequences induced by depletion or knock-out of the NRG1 gene or exogenous NRG1 injection have been extensively studied over the last decades. Neuregulins can function as a ligand for ErbB4, an EGF receptor. NRG1/ErbB signaling has been implicated in GABAergic circuit formation, axon myelination, and synaptic plasticity (Ghashghaei et al., 2006;Li et al., 2007;Woo et al., 2007;Fazzari et al., 2010;Abe et al., 2011;Ting et al., 2011;Yin et al., 2013). Furthermore, type III NRG1 plays a role in morphological development of cortical neurons independently of ErbB kinase activity (Chen et al., 2008;Chen et al., 2010). Although NRG2/ErbB4 signaling has been implicated in the proliferation of neural stem cells and neuroblasts in the subventricular zone (Ghashghaei et al., 2006), little is understood about whether it plays a role in synaptogenesis of newborn neurons. In general, GABAergic synaptogenesis precedes glutamatergic synaptogenesis in the early developmental stage of newborn neurons. It has been demonstrated that depolarization induced by GABAergic synaptic inputs in immature neurons is important for AMPA receptor insertion to nascent glutamatergic synapses that express only NMDA receptors (Akerman and Cline, 2006;Wang and Kriegstein, 2008), suggesting that GABAergic synaptogenesis is a prerequisite for maturation of glutamatergic synapses in immature neurons. Given that NRG1/ErbB4 signaling plays a critical role in GABAergic synapse formation (Fazzari et al., 2010), it should be differentiated whether the effects of neuregulin signaling on glutamatergic synapse maturation are direct or secondary to its effect on GABAergic synaptogenesis. Whereas CNS-specific knock-out of ErbB2/4 impaired dendritic spine maturation (Barros et al., 2009), a recent study has shown that conditional knock-out of ErbB4 in neocortical pyramidal neurons caused no effect on excitatory synapses (Fazzari et al., 2010). Therefore, cell- and developmental-stage-specific gene manipulation is essential to elucidate biological functions exerted directly by neuregulins. NRG2 is the major type of neuregulin expressed in hippocampal dentate granule cells (GCs). We set out to address the question of whether NRG2 plays any cell-autonomous role in the development of newborn GCs, especially during two critical developmental periods when GABAergic and glutamatergic synapses are established. To this end, we depleted NRG2 at different developmental stages of newborn GCs using a tetracycline-inducible ( Tet-On) expression system (Gossen and Bujard, 1992). Here, we show that NRG2 plays a central role in GABAergic synaptogenesis and glutamatergic synapse maturation in newborn GCs via forward and reverse signaling pathways, respectively. Materials and Methods Organotypic slice culture. All experiments were performed with the approval of the animal experiment ethics committee at the Seoul National University College of Medicine. The entorhino-hippocampi of postnatal day 6–9 (P6–P9) Sprague Dawley rat pups (Charles River) of either sex were cultured by using previously described membrane interface techniques (Lee et al., 2012b). Briefly, the transverse hippocampal slices (thickness, 300 μm) were obtained with vibratome (DTK-1000 ZERO 1; Dosaka) and carefully placed on the surface of 30 mm porous (0.4 μm) membrane insert (Millicell-CM; Millipore) set in a 6-well plate on 1 ml of culture medium. Because deprivation of major synaptic inputs of GCs may alter the responsiveness to NRG2, we took great care to preserve the perforant pathway and entorhinal cortex in the organotypic culture. The culture medium was a mixture of 50% minimum essential medium, 25% horse serum, 24% Eagle's buffered salt solution, and 1% penicillin–streptomycin (PS). Glucose was added to reach a final concentration of 36 m m. To examine the role of NRG2 in synaptogenesis, we infected the slices with retrovirus immediately after transferring slices on membrane. After 4 d in culture, the medium was changed to serum-free Neurobasal medium with 2% B-27 supplement, 1% GlutaMAX-I, 1% PS, and 5 m mglucose added. Culture medium was changed every other day. Slice cultures were maintained at 36°C in a 5% CO 2humidified incubator. All media and reagents for organotypic culture were purchased from Invitrogen. Retroviruses, conditional knock-down, and conditional transgene expression. Newborn GCs were labeled using vesicular stomatitis virus glycoprotein pseudotyped recombinant retrovirus encoding EGFP and microRNA. These two genes were cloned downstream of long terminal repeat (LTR) promoter and a Tet-Onpromoter, respectively, in the same Moloney murine leukemia virus (MMLV)-derived retroviral vector (seeFig. 1 C). Therefore, the expression of miR30-based short-hairpin RNA (shRNAmir) was under the control of the tetracycline-inducible expression system (Gossen and Bujard, 1992). We targeted the shRNAmir by inserting the partial sequence of NRG2 (5′-GGTGTGTCACTCGCCTGCTACT-3′) into the backbone of miR30, an endogenous microRNA (Dickins et al., 2005). This will be referred to as “shNRG2mir.” The fluorescent protein mCherry-targeting sequence (5′-GAACATCAAGTTGGACATCACC-3′) was used for nontargeting control of shRNAmir (shNTmir).Figure 1 Cshows the schematic diagram for the expression cassette of EGFP, reverse tetracycline transactivator 3 (rtTA3), and shRNAmir. We engineered retroviral construct by inserting this cassette (2.6 kb) between the LTRs of EGFP-CL retroviral vector (Lee et al., 2012a). For rescue experiments, C-terminal myc-tagged intracellular domain of NRG2 (NRG2-ICD) was cloned downstream of the Tet-Onpromoter in the TagRFP-CL retroviral vector, where TagRFP was substituted instead of the EGFP in EGFP-CL (seeFig. 7 A1). To package retroviruses, each of these plasmid DNAs were transfected into 293GPG packaging cells using Lipofectamine 2000 (Invitrogen). Supernatant containing viral particles was harvested at 3–10 d after transfection and centrifuged at 6000 × gfor 16 h at 4°C. The viral pellet was then resuspended in 1/500 of the original volume of DPBS (Invitrogen). The dentate gyrus of a hippocampal slice culture was infected with the virus solution (0.3 μl) within 5 min after placement of hippocampal slices on the membrane insert as described previously (Namba et al., 2007;Lee et al., 2012a). For conditional knock-down or conditional expression, supplement of doxycycline (Dox, 2 μg/ml) in culture medium was started 2 d before the time that knock-down or overexpression was desired. Single-cell RT-PCR. Hippocampal newborn GCs were labeled by retrovirus encoding EGFP. To obtain total mRNA of newborn GC, whole-cell patch-clamp was attained on the EGFP-expressing soma of a dentate GC at 5–6 days postinfection (dpi). Pipette solution was composed of the following (in m m): 140 K-gluconate, 5 di-Tris-phosphocreatin, 5 NaCl, 4 MgATP, 0.4 Na 2GTP, 15 HEPES, 2.5 Na-pyruvate, pH 7.3 adjusted with KOH, together with 10% RNaseOUT (v/v), a recombinant ribonuclease (RNase) inhibitor (Invitrogen). After rupturing the giga-ohm-sealed cell membrane patch, cytosolic contents were sucked into the glass pipette by applying negative pressure. To prevent the pipette solution from being contaminated by RNase, the whole-cell patch mode was converted to outside-out configuration by gently pulling up the pipette away from the cell. The pipette tip containing the cytosolic contents was broken into a 0.2 ml PCR tube with 7 μl of premixture containing 1 μl of dNTP (25 m meach), 1 μl of 0.1 mDTT, 1 μl of random hexamer oligonucleotides as primers, and 4 μl of 5× first-strand buffer (Invitrogen). Then, 1 μl of SuperScript III reverse transcriptase (200 U/μl; Invitrogen) and RNase-free water were added to make a total volume of 20 μl of solution. All subsequent steps for synthesizing cDNA followed the user manual of SuperScript III reverse transcriptase. Random primers were annealed to mRNA template by incubating at 65°C for 5 min. cDNA was synthesized under the following conditions: 25°C for 5 min and 50°C for 60 min. The reaction was terminated by heating at 70°C for 15 min. Synthesized single-strand cDNA was amplified by PCR with GoTaq Green Master Mix (Promega) and the following primers: NRG2β forward 5′-CAGCAGTTCGCAATGGTCAACT-3′, reverse 5′-GTAATCTGCCATCTGGATCTCC-3′; β-actin forward 5′-GCCGCATCCTCTTCCTCCCTG-3′, reverse 5′-GGGGCCGGACTCATCGTACTC-3′. The PCR products were examined by standard agarose gel electrophoresis. In situ hybridization and immunohistochemistry. For in situhybridization, a postnatal week 4 (PW4) rat was perfused by PBS and subsequently fixed with 4% paraformaldehyde (PFA). Brain was postfixed with 4% PFA and then dipped with 30% sucrose in PBS. After sinking, the brain was embedded in optimum cutting temperature compound and sectioned serially into 16 μm sections. The sequence of the ErbB4 mRNA target probe was manufactured by Advanced Cell Diagnostics. A probe targeting the bacterial gene dapB was used as a negative control. In situhybridization of ErbB4 mRNA was performed on sectioned samples according to the RNAscope multiplex fluorescence kit. Briefly, tissue sections were dried at 50°C for 30 min and then fixed with 4% PFA for 15 min; dehydrated by 50%, 70%, and 100% ethanol gradually for 5 min; and pretreated with the probe kit before hybridization. After hybridization with ErbB4 mRNA at 40°C for 2 h, the RNAscope multiplex fluorescence kit was used for signal amplification. Fluorescence detection was performed using Atto 550 (red fluorescence). Immunohistochemistry was performed using a primary antibody against the doublecortin (DCX, 1:500; Santa Cruz Biotechnology) for 1 h at 37°C. Alexa Fluor 488-conjugated donkey anti-rabbit IgG was applied for 30 min (1:500) with Hoechst 33343 for staining nuclei. The sections were washed three times and mounted. Western blotting. HEK293 cells were seeded in 100 mm culture dishes at ∼70% confluence and transfected with C-terminally myc-tagged NRG2 (NRG2-myc) plus shNRG2mir or plus shNTmir (as control) using the calcium phosphate protocol. Mouse cDNA of NRG2 was purchased from Origene (catalog #MR217926). After culture for 48 h, the cells were washed twice with PBS (pH 7.4) solubilized in ice-cold lysis buffer containing 20 m mTris (pH 7.4 adjusted with HCl) 140 m mNaCl, 10% sucrose, 1 m mEDTA, 1 m mNa 3VO 4, 1% NP-40 (v/v), and 0.2% protease inhibitor mixture. Cell lysates were mixed with equal volume of the 2× SDS sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The resulting blots were blocked for 1 h in PBS plus 0.1% Triton X-100 (0.1% PBST) containing 5% skim milk (blocking solution) and incubated overnight at 4°C with specific primary antibodies: mouse monoclonal anti-c-myc (1:1000; Cell Signaling Technology) or mouse monoclonal anti-β-actin (1:5000; Santa Cruz Biotechnology) as loading controls. Immunoblots were washed with washing buffer containing 1% NP-40 and 0.1% SDS in PBS for 1 h, then with PBS twice each for 30 min at room temperature. Blots were blocked in blocking solution for 30 min and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000; Jackson ImmunoResearch). After washing with washing buffer and PBS, detection was performed using enhanced chemiluminescence reagent (GE Healthcare Bioscience). The membranes were then exposed to x-ray films (Agfa Gevaert). Immunohistochemistry. Expressions of NRG1 and NRG2 in the hippocampal slice cultures and tetracycline-inducible knock-down of NRG2 or expression of myc-tagged NRG2-ICD were confirmed by immunostaining of endogenous NRG1 and NRG2 or myc in organotypic cultured hippocampal slices. Dox treatment was started 3–5 d before fixation. Slices were fixed overnight at 4°C by submersion in 4% PFA dissolved in PBS. The next day, after 3 washes with PBS containing 0.3% Triton X-100 (0.3% PBST), slices were incubated in blocking solution (5% donkey serum in 0.3% PBST) for 1 h at room temperature and then with rabbit polyclonal anti-NRG2 IgG (1:500; Millipore; seeFig. 1 E) or mouse monoclonal anti-NRG1 IgG (1:200; Millipore; seeFig. 1 F) or mouse monoclonal anti-myc (1:2000; Cell Signaling Technology; seeFig. 7 A2) diluted in blocking solution overnight at 4°C. After 3 washes in 0.3% PBST for 10 min, slices were incubated with Cy3-conjugated goat anti-rabbit (1:400; Invitrogen), Alexa Fluor 647-conjugated donkey anti-mouse (1:200; Jackson ImmunoResearch), or FITC-conjugated donkey anti-mouse (1:200; Jackson ImmunoResearch) diluted in blocking solution for 1 h at room temperature. Finally, the slices were washed 3 times in 0.3% PBST store in PBS until confocal imaging was performed. Electrophysiology. Postsynaptic current (PSC) was recorded in EGFP-positive newborn GCs using whole-cell patch-clamp techniques while perfusing a slice with artificial CSF (aCSF). The aCSF contained the following (in m m): 124 NaCl, 26 NaHCO 3, 3.2 KCl, 2.5 CaCl 2, 1.3 MgCl 2, 1.25 NaH 2PO 4, and 10 glucose, pH 7.4 adjusted by saturating with carbogen (95% O 2, 5% CO 2). PSC was evoked by stimulation (200 μs duration; 1–10 V intensity) of axons passing through the inner molecular layer (IML) of hippocampal dentate gyrus using a concentric bipolar electrode with 30 s interstimulus interval. The stimulation electrode was placed at least 200 μm apart laterally from the cell under recording to avoid direct stimulation of a recorded cell. To isolate GABAergic PSCs (GPSCs), the membrane potential was held at 0 mV, which is the reversal potential of excitatory PSCs (EPSCs). The internal pipette solution for recording GPSCs contained the following (in m m): 140 K-gluconate, 5 di-Tris-phosphocreatin, 5 NaCl, 4 MgATP, 0.4 Na 2GTP, 15 HEPES, 2.5 Na-pyruvate, 1 EGTA, pH 7.3 adjusted with KOH. For isolation of EPSCs, we could not use a GABA Areceptor blocker because it induces epileptiform activity. Instead, GPSCs were blocked intracellularly using a CsF-based pipette solution containing the following (in m m): 140 CsF, 2 MgCl 2, 10 HEPES, 1 EGTA (Marchionni and Maccaferri, 2009;Schwartz et al., 2011), and a Cl −channel blocker, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 1 m m) (Hollrigel et al., 1998), pH 7.3 adjusted with CsOH. AMPA receptor- and NMDA receptor-mediated EPSCs (AMPA-EPSCs and NMDA-EPSCs) were recorded at holding potentials of −60 and +40 mV, respectively. The amplitudes of GPSCs and AMPA-EPSCs were measured at their peak; the amplitude of NMDA-EPSCs was measured at 50 ms after stimulation to exclude AMPA-EPSC contribution. The paired-pulse ratio (PPR) was calculated from the mean amplitude of the second PSCs divided by the mean amplitude of the first PSCs. The interpulse interval for GPSCs and NMDA-EPSCs was 500 ms and that of AMPA-EPSCs was 30 ms. The AMPA-to-NMDA EPSC ratio (AMPA/NMDA ratio) was calculated by dividing the peak amplitude of AMPA-EPSCs by the amplitude of NMDA-EPSCs measured at 50 ms, similar to the procedure described byMierau et al. (2004). To measure monosynaptic GPSCs, monopolar stimulation electrode (a glass pipette filled with aCSF) was placed ∼100 μm lateral from the recording position. Monosynaptic responses were evoked by extracellular stimulation (200 μs duration; 1–25 V intensity) with 30 s interstimulus interval in the presence of 20 μ mCNQX and 50 μ mAPV to block glutamatergic transmission. The amplitudes of monosynaptic GPSCs compared inFigures 3 Cand6 Bwere obtained from a minimal stimulation protocol. The first criterion for the minimal stimulation is an abrupt appearance of synaptic responses as a gradual increase in the stimulation intensity. There should be a distinct threshold for such all-or-none activation of synaptic responses. Moreover, further increase in the stimulation intensity should not change the synaptic responses by ∼20% increase in stimulus intensity from the threshold (Dvorzhak et al., 2013). Typical voltage intensities required for minimal stimulation ranged between 5 and 12 V. In vivo experiments. P16–P22 SD rats were anesthetized by inhalation of ∼1–4% isoflurane, shaved, and then placed in a stereotaxic frame (Neurostar; Stoelting). All surgical procedures were performed under aseptic conditions. Retrovirus encoding EGFP plus Tet-On/shNRG2mir was infused through a glass micropipette which was prepared using a vertical puller (Narishige PC-10) and a pipette beveler (beveling angle, 30°; Narishige EG-4). Virus solution of 0.5 μl was injected at a rate of 0.15 μl/min by a 10 μl Hamilton syringe attached to a digital microinjector (Stoelting). The injection coordinates were −4.8 mm A/P, ± 4.4 mm M/L, −4.6 mm D/V from the bregma. The glass pipette was slowly removed after holding it in the injection site for 3 min to allow for the viral diffusion. After suturing, the rats were returned to the cages and lidocaine/antibiotics ointment was applied over the sutured region. Rats were given drinking water containing 2 mg/ml Dox plus 2% sucrose (Dox-containing water) or 2% sucrose alone (Dox-free water) from 5 dpi or from 9 dpi. The drinking water was changed every other day. Acute hippocampal slices (thickness, 300 μm) were prepared from 12–21 dpi (P28–P40) rats as described inLee et al. (2007). Drugs. All chemicals were purchased from Sigma-Aldrich except CNQX and bicuculline methiodide, which were from Tocris Bioscience; APV, which was from Abcam; and AG-1478, which was from Calbiochem. Stock solutions were stored at −20°C and diluted to the desired concentration in aCSF just before use. Anti-c-erbB4 Ab-3 mouse monoclonal antibody (MS-304) was purchased from Thermo Scientific. Sholl analysis and measurement of total dendritic length. EGFP fluorescence expressed in newborn GCs were imaged using a confocal laser scanning microscope (FV300; Olympus) and a 40× water-immersion objective. For analysis of dendritic morphology, 3D reconstructions were made using the Simple Neurite Tracer plugin in the ImageJ-based image processing package Fiji (Longair et al., 2011). The dendritic complexity and length were quantified by Sholl analysis plugin and tracing algorithms in NeuronJ plugin, respectively. For Sholl analysis, the concentric circles were drawn every 10 μm from the first concentric circle, which had a diameter 5 μm encircling the soma, and the numbers of branch intersections were counted in each circle. Data analysis. Data were analyzed using Igor-Pro (version 6.1; WaveMetrics) or IBM SPSS statistics version 21.0 software. Statistical data are expressed as mean ± SEM and nindicates the number of cells studied. If not mentioned otherwise, the statistical significance of differences between two experimental conditions was evaluated using Student's ttest. Comparisons between input–output curves of synaptic currents and between results of Sholl analysis were analyzed using repeated-measures (RM) one-way ANOVA (seeFigs. 3 A1,4 A,5 B,7 D2). To compare between cumulative lengths of neurites, the Kolmogorov–Smirnov (K-S) test was used (seeFigs. 5 C,7 D3). Differences were considered as significant when p< 0.05 (n.s. indicates no statistical significance; * p< 0.05; p< 0.01; * p< 0.001). Results Confirmation of NRG2 expression in newborn GCs and conditional knock-down strategy of NRG2 Although NRG2 is highly expressed in the GC layer of dentate gyrus of adult hippocampus (Anton et al., 2004;Longart et al., 2004), it is not clear whether newborn GCs express NRG2. To address this question, newborn GCs from cultured rat hippocampal slice were labeled with EGFP-expressing retrovirus and then their mRNA was analyzed by single-cell RT-PCR (see Materials and Methods for details). There are two alternative splicing isoforms of NRG2, α and β, and only the β isoform is capable of stimulating ErbB4 (Hobbs et al., 2002). The cytoplasm of EGFP-positive single newborn GC was aspirated using a whole-cell patch pipette (Fig. 1 A1) and mRNA therein was amplified using a primer pair specific for an ErbB4-stimulating isoform, NRG2β. NRG2β transcript was detected in 7 of 8 newborn GCs and β-actin, used as positive control, was detected in all samples (Fig. 1 A2). Figure 1. Tests for expression of NRG1, NRG2, and ErbB4 in hippocampal newborn GCs and for shRNAmir-mediated depletion of NRG2. A1, Extraction of cytoplasm for single-cell RT-PCR. Left, Retrovirus-infected newborn GCs were identified by EGFP fluorescence at 5–6 dpi. Right, Cytoplasm was sucked into the whole-cell pipette with negative pressure. Arrowheads, EGFP-positive newborn GC; arrow, patch pipette. Scale bar, 10 μm. A2, Representative example for single-cell RT-PCR. Bands for NRG2β and β-actin (as a positive control) were detected at the predicted size of 282 and 414 bp, respectively. The leftmost lane is a size marker. B1, Immunostaining of endogenous NRG1 in the hippocampal slice culture (7 dpi) shows that NRG1 signal is weaker in dentate gyrus (DG) than in CA1 and CA3 regions (top). Bottom, Corresponding DIC image. Scale bar, 300 μm. B2, Double staining of ErbB4 transcripts (magenta) and DCX (green). Boxed regions of DG, CA3, and CA1 in top left panel are magnified images. ErbB4 signal was negative in the DCX +cells (arrow) in the DG. Note that there are many ErbB4 fluorescent spots in DCX −ErbB +cells (arrowhead). Hoechst33342 (blue) was used for nuclear counterstaining. Scale bar, 40 μm. C, Schematic of the tetracycline-inducible shRNAmir expression cassette. EGFP and rtTA3 are constitutively expressed under the control of promoters, the 5′ LTR, and human ubiquitin-C promoter (pUbiC), respectively. The rtTA3 binds to TRE and activates the transcription of shRNAmir in the presence of doxycycline. Ψ, Packaging signal; rtTA3, reverse tetracycline transactivator; TRE, tetracycline response element; pCMVmin, mini-CMV promoter; WPRE, wood-chuck hepatitis B posttranscriptional regulatory element. D, shNRG2mir was cotransfected with NRG2-myc into HEK293 cells. When Dox (2 μg/ml, 48 h) was applied to HEK293 cells, shNRG2mir almost depleted NRG2-myc, but shNTmir did not. The blot of β-actin is shown as a loading control. TF, Transfection. E1, F1, NRG2 and NRG1 immunofluorescence (magenta) of the dentate GC layer (7 dpi) in organotypic cultures infected with retrovirus encoding EGFP plus Tet-On/shNRG2mir. Slice cultures were treated (Tx.) with Dox started at 4 dpi (bottom) or untreated (top) and then fixed and immunostained at 7 dpi. Somata of EGFP-positive GCs are indicated by arrowheads. Scale bar, 20 μm. E2, F2, Mean values for the NRG2 and NRG1 immunofluorescence intensity in the EGFP-positive regions of Dox-treated and untreated slices were quantified at 7 or 14 dpi. Dox treatment was from 4–7 dpi or from 10–14 dpi, respectively. We further examined the expressions of NRG1 and NRG2 in hippocampal slice cultures at 7 and 14 dpi (equivalent to PW2 and PW3 in vivo, respectively) using immunohistochemistry. Consistent withLongart et al. (2004), the NRG1 immunosignal was weaker in the dentate gyrus compared with the CA1 and CA3 regions (Fig. 1 B1), suggesting that NRG2 may be a major ligand for ErbB4 in the dentate gyrus. Next, we investigated whether newborn GCs express the NRG2 receptor ErbB4. It has been noted that ErbB4 is expressed exclusively in GABAergic interneurons, at least in the neocortex and hippocampus (Vullhorst et al., 2009;Fazzari et al., 2010). Nevertheless, the expression of ErbB4 in newborn GCs remains unknown. To determine whether immature GCs express ErbB4, we doubly stained the hippocampus for ErbB4 transcripts and DCX. The former was stained by in situhybridization and the latter, which is a marker of immature GCs (Rao and Shetty, 2004), was stained by immunohistochemistry. This double staining revealed that the signal of ErbB4 transcripts is noise level in most DCX-immunoreactive cells of the dentate gyrus (0.59 ± 0.14, n= 180, 3 rats,Fig. 1 B2). In contrast, some cells scattered in the dentate gyrus showed significantly many ErbB4 fluorescent spots (44.49 ± 3.17, n= 45) that were presumably inhibitory interneurons. Therefore, it is unlikely that NRG2 on newborn GCs act as an autocrine ligand. To study the role of NRG2 in GABAergic and glutamatergic synaptogenensis on newborn GCs, we tried to knock down the NRG2 transcripts specifically in newborn GC using retrovirus. Because NRG2/ErbB4 signaling regulates the proliferation of neuroblasts in the subventricular zone (Ghashghaei et al., 2006), constitutive knock-down of NRG2 may suppress the proliferation of neural progenitor cells in the dentate gyrus. Therefore, we designed a cassette encoding shNRG2mir under the control of the Tet-Onpromoter (Fig. 1 C; see Materials and Methods for details;Gossen and Bujard, 1992) and inserted it into the MMLV retroviral vector. This retroviral construct enabled us to control the period of NRG2 knock-down by treatment with Dox (2 μg/ml). In addition, the cassette encodes the EGFP gene downstream of the constitutively active LTR promoter, so we could label newborn GCs infected by the retrovirus regardless of Dox treatment (Fig. 1 C). The knock-down efficiency of shNRG2mir was tested in HEK293 cells heterologously expressing NRG2-myc together with shNRG2mir or shNTmir. When the HEK293 cells were treated with Dox (2 μg/ml) for 48 h, shNRG2mir, but not shNTmir, almost depleted NRG2-myc. In untreated cells, neither shNTmir nor shNRG2mir depleted NRG2-myc (Fig. 1 D). Next, to test whether shNRG2mir efficiently depletes endogenous NRG2 in newborn GCs, we compared immunofluorescence of NRG2 in EGFP-positive newborn GCs at 7 dpi grown in the rat hippocampal slice cultures under control conditions and those treated with Dox from 4 dpi. The immunoreactivity of NRG2 was significantly lower in the Dox-treated newborn GCs compared with the untreated GCs (Fig. 1 E, untreated, 940.97 ± 79.87, n= 9 vs Dox-treated, 558.60 ± 21.22, n= 27, p< 0.001), whereas NRG1 immunoreactivity was not affected by shNRG2mir (Fig. 1 F). Repeating the same experiments for newborn GCs at 14 dpi that were untreated or treated with Dox from 10 dpi, we obtained similar results (Fig. 1 E2, untreated, 822.69 ± 53.33, n= 11 vs Dox-treated, 466.55 ± 35.32, n= 22, p< 0.001), indicating that shNRG2mir efficiently depletes endogenous NRG2 in newborn GCs and that knock-down can be induced by Dox treatment. Developmental changes of postsynaptic currents in newborn GCs under control conditions Before studying the effect of NRG2 depletion, we examined the normal developmental increase in the PSC of newborn GCs. Newborn GCs grown in rat hippocampal slice cultures were identified by expression of EGFP (Fig. 2 A). At different dpi's, the IML of the hippocampal dentate gyrus was stimulated to evoke GPSCs, AMPA-EPSCs, and NMDA-EPSCs (Fig. 2 B). The K-gluconate-based pipette solution containing 5 m mCl −was used for recordings of GPSCs. In hippocampal dentate gyrus, glutamatergic afferent fibers, such as perforant pathways and hilar mossy cell axons, are mostly confined to the molecular layer and run in parallel with the GC layer (Buckmaster et al., 1992;Witter, 2007;Chancey et al., 2014), whereas GABAergic axon fibers are highly branched and convoluted over all three layers of dentate gyrus (Soriano and Frotscher, 1989;Bartos et al., 2001;Markwardt et al., 2011). Because a bipolar stimulation electrode was placed at least 200 μm laterally apart from the recorded newborn GC, the electrode is more likely to stimulate glutamatergic afferent fibers rather than GABAergic ones. Consistently, putative GPSCs, which were recorded as an outward PSC at the reversal potential of EPSCs (0 mV), were inhibited not only by bicuculline (20 μ m), a GABA Areceptor blocker, but also by CNQX (20 μ m), an AMPA receptor blocker (Fig. 2 C), implying that the GPSCs evoked by IML stimulation are caused by bisynaptic feedforward GABAergic inputs (Fig. 2 B). Figure 2. Developmental changes of GPSCs and EPSCs in newborn GCs. A, Dentate gyrus of a hippocampal organotypic slice culture at 10 dpi. Newborn GCs infected by retroviruses expressed EGFP. The borders between the GC layer (GCL) and hilus or between the GCL and the IML are marked by dashed lines. Scale bar, 50 μm. B, Schema for excitatory and feedforward inhibitory synapses innervating a newborn GC. Stimulating electrode was located in IML (blue double lines). GPSCs or EPSCs were recorded from EGFP-positive newborn GCs in GCL using whole-cell patch-clamp techniques. Stim, Stimulation electrode; Rec, recording pipette; IN, interneuron. C, Representative GPSCs recorded in newborn GCs. Note that feedforward GPSCs were blocked not only by bicuculline (20 μ m, C1) but also by CNQX (20 μ m, C2). Scale bar, 200 pA and 200 ms. D, Representative EPSCs recorded at the holding potentials of −60 mV ( D1) and at +40 mV ( D2) in newborn GCs. Scale bar, 500 pA and 200 ms. D1, AMPA-EPSCs were abolished by CNQX (20 μ m). D2, When APV (50 μ m) was bath-applied to the slice, NMDA-EPSCs were and the outward AMPA-EPSCs were unaffected. The amplitude of NMDA-EPSCs was measured 50 ms after the stimulation (red dashed line and arrow in the inset). Insets, Traces in an expanded time scale. Scale bars, 500 pA and 50 ms. E, Averaged input–output curves of GPSCs (top), AMPA-EPSCs (middle), and NMDA-EPSCs (bottom) from newborn GCs. The amplitudes of evoked GABAergic or glutamatergic PSCs were plotted as a function of stimulus intensity at different dpi's. F, Developmental changes in amplitudes of GPSCs, AMPA-EPSCs, and NMDA-EPSCs recorded in newborn GCs grown in organotypic slice cultures under control conditions. All PSCs were evoked by stimulation of IML at 10 V. The age of GCs are shown as dpi, which represents the number of days elapsed from the day when the slice was infected by EGFP-encoding retrovirus. The number on each symbol indicates number of cells studied ( n). AMPA-EPSCs and NMDA-EPSCs were recorded using a CsF-based pipette solution containing 1 m mDIDS to block Cl −current (see Materials and Methods for details). AMPA-EPSCs were evoked at a holding potential of −60 mV and completely attenuated by CNQX (20 μ m) (Fig. 2 D1). NMDA-EPSCs were evoked at a holding potential of +40 mV and inhibited by 50 μ mAPV, an NMDA receptor blocker (Fig. 2 D2). The input–output curves for the three kinds of PSCs in newborn GCs were obtained by plotting the amplitudes of evoked PSCs as a function of stimulus intensity.Figure 2 Eshows mean input–output curves of GPSCs, AMAP-EPSCs, and NMDA-EPSCs at different dpi's of newborn GCs. Because most of these curves reached a plateau at the stimulation voltage of 6 or 7 V, the mean values of GSPCs and EPSCs at 10 V were collected and plotted as a function of the age of newborn GCs denoted as dpi (Fig. 2 F). This plot represents the normal time courses for the developmental increases of synaptic inputs to newborn GCs grown in slice cultures. The evoked GPSCs distinctly appeared in newborn GCs as early as 6 dpi and their amplitude was steeply increased until 10 dpi. After 10 dpi, the evoked GPSC amplitude did not further increase. Conversely, AMPA-EPSCs were negligible until 11 dpi and then continuously increased at least to the oldest age we studied (∼16 dpi). NMDA-EPSCs were observed earlier than AMPA-EPSCs and increased gradually. Because the increase rate of NMDA-EPSCs was less steep than that of AMPA-EPSCs, the AMPA/NMDA ratio increased developmentally (seeFigs. 2 F,4 C). Expression of NRG2 is essential for GABAergic synapse formation but dispensable for maintenance in newborn GCs We studied whether depletion of NRG2 has any effect on the developmental changes of GPSCs.Figure 1 Eshows that NRG2 is depleted to ∼50% in the 3 d after starting Dox treatment. Therefore, we started to treat cultured hippocampal slices with Dox from 4 dpi and maintained the Dox treatment until the day of the experiment to deplete NRG2 during the GPSC-increasing phase at 6–9 dpi. We obtained input–output curves for mean amplitudes of GPSCs recorded from control and Dox-treated newborn GCs at different dpi's. Typically, GPSCs started to be evoked by stimulation of IML at 3 V and their amplitude became steady at 6 V and higher stimulation voltages. Amplitudes of GPSCs were significantly lower in Dox-treated newborn GCs than those in control conditions at both ages (Fig. 3 A1; control vs Dox, F (1,35)= 12.03, p= 0.001 at 7 dpi; F (1,15)= 24.04, p< 0.001 at 11 dpi, RM one-way ANOVA). The effect of NRG2 depletion on GPSCs is summarized inFigure 3 A2, which displays the mean amplitude of GPSCs evoked at 10 V as a function of dpi. This plot demonstrates that the depletion of NRG2 significantly suppresses the developmental increase of GPSCs. Figure 3. NRG2 is essential for GABAergic synapse formation in newborn GCs. A1, Averaged input–output curves of GPSCs in control (black) and NRG2-depleted (red, Dox-treated from 4 dpi) newborn GCs at 7 dpi (open circles, dotted line) or at 11 dpi (solid circles, solid line). For both GPSCs at 7 and 11 dpi, amplitudes were significantly lower in Dox-treated GCs (control vs Dox; 7 dpi, F (1,35)= 12.03, p= 0.001; 11 dpi, F (1,15)= 24.04, p< 0.001 by RM one-way ANOVA). A2, Effects of NRG2 knock-down on the developmental changes of feedforward GPSCs in newborn GCs. To deplete NRG2 hippocampal slices were treated (Tx.) with Dox from 4 dpi. GPSCs in Dox-treated GCs were significantly lower than control cells as early as 7 dpi. A3, Effects of NRG2 knock-down on GPSCs after the critical period of GABAergic synapse formation (6–9 dpi). The slice cultures were treated with Dox from 7 dpi (blue) or 10 dpi (purple). GPSCs of the treated slice were not significantly different from the untreated control slices at all ages ( p> 0.1). In A2and A3, mean amplitudes of GPSCs evoked by 10 V stimulation of IML were plotted as a function of dpi. A4, Mean amplitudes of GPSCs in newborn GCs older than 9 dpi, at which GPSCs reached a plateau under different Dox treatment conditions. B1, Monosynaptic GPSCs were evoked by minimal stimulation in the presence of AMPA and NMDA receptor antagonists using a monopolar electrode positioned 30–100 μm apart from the soma of a newborn GC. Rec, Recording pipette; IN, interneuron. B2, Representative trace of monosynaptic GPSCs, which was completely blocked by 20 μ mbicuculline, a GABA Areceptor blocker. Scale bar, 50 pA and 100 ms. B3, Representative input–output curve for monosynaptic GPSCs in newborn GCs. As the stimulation intensity was gradually increased from 1 V, the synaptic responses were all-or-none at a distinct threshold (6 V) and were kept stable for further increase by 5 V from the threshold. Such stable range of stimulation intensity was adopted as the voltage intensity for minimal stimulation (marked with yellow). C, Mean amplitudes of monosynaptic GPSCs in newborn GCs grown in slice cultures ( ex vivo, C1) or acute slices ( in vivo, C2). The starting dates for Dox treatment or feeding (Dox Tx.) are indicated in the abscissa. The recording dates are indicated above the bar graphs. Monosynaptic GPSCs were suppressed by early Dox Tx (from 4 or 5 dpi), but not by late Dox Tx (from 7 or 9 dpi), in both ex vivoand in vivoexperiments. The above results indicate that NRG2 is essential for developmental increase in GABAergic inputs at 6–9 dpi, which may be a critical period for GABAergic synapse formation under our experimental conditions (Fig. 2 F). To address the question of whether NRG2 is necessary after this critical period, the hippocampal slice cultures were treated with Dox from 10 dpi and GPSCs in the Dox-treated newborn GCs were examined at 13 or 14 dpi. As shown inFigure 3 A3, Dox treatment at this age had no significant effect on GPSC amplitudes, arguing against a role of NRG2 in maintenance of GABAergic synapses (control vs Dox-treated from 10 dpi, p> 0.1). To confirm this notion, we started the Dox treatment earlier. The Dox treatment starting at 7 dpi might be sufficient for depleting NRG2 just after the critical period (9 or 10 dpi) because knock-down of NRG2 occurs, at the latest, 2 or 3 d after starting Dox treatment (Fig. 1 E). This earlier start of Dox treatment also had no significant effect on GPSCs measured in newborn GCs between 9 and 14 dpi (Fig. 3 A3, control vs Dox-treated from 7 dpi, p> 0.1).Figure 3 A4shows the age dependence of NRG2 knock-down effects on the GPSCs. Mean values of GPSC amplitudes were measured in newborn GCs older than 9 dpi, when GPSC amplitudes reached a plateau. Dox treatment from 4 dpi, but not from 7 or 10 dpi, significantly reduced the GPSC amplitude (Fig. 3 A4, control, 0.950 ± 0.054 nA, n= 65; Dox-treated from 4 dpi, 0.306 ± 0.032 nA, n= 27, p< 0.001; Dox-treated from 7 dpi, 0.905 ± 0.054 nA, n= 39, p= 0.577; Dox-treated from 10 dpi, 0.889 ± 0.071 nA, n= 14, p= 0.611). These results demonstrate that NRG2 is necessary for GABAergic synapse formation during the critical period (6–9 dpi), but, after this critical period, it is dispensable for the maintenance of GABAergic synapses in newborn GCs. Next, we verified the above notions in monosynaptic GPSCs in slice cultures ( ex vivo) and in vivo. Monosynaptic GPSCs were evoked by local stimulation of GC layer ∼70 μm apart from the recorded cell in the presence of AMPA and NMDA receptor blockers in the bath solution (Fig. 3 B1) and recorded as outward PSCs at the holding potential of 0 mV. This outward PSC was completely abolished by bicuculline, indicative of monosynaptic GPSCs (Fig. 3 B2). A minimal stimulation protocol was used to evoke monosynaptic GPSCs (Fig. 3 B3, see Materials and Methods for details). Consistent with the results of feedforward GPSCs, the monosynaptic GPSCs recorded at 14 or 15 dpi ex vivowere suppressed by early Dox treatment, but not by late Dox treatment (Fig. 3 C1). For in vivoexperiments, newborn GCs were labeled by stereotaxic injection of the same retrovirus used in ex vivoexperiments into the hippocampal dentate gyrus of rats (16–22 d old). We studied monosynaptic GPSCs in the acute slice of such rats after feeding them with Dox-free or Dox-containing (2 mg/ml) water. Because maturation of newborn GCs is faster at a younger age (Overstreet-Wadiche et al., 2006a), Dox feeding started 1 or 2 d later than ex vivo, that is, at 5 and 9 dpi. The amplitude of GPSCs recorded 10 d later were significantly suppressed by Dox feeding from 5 dpi, but not by Dox feeding from 9 dpi, confirming the results of ex vivoexperiments (Fig. 3 C2). NRG2 is essential for functional maturation of glutamatergic synapses and dendritic growth in newborn GCs During the early postnatal week, GABAergic inputs exert depolarizing influences on newborn neurons. Perturbation of such GABA-induced depolarization by overexpression of KCC2 or knock-down of NKCC1 blocks normal development of AMPA receptor-mediated neurotransmission (Akerman and Cline, 2006;Ge et al., 2006), suggesting that GABAergic synaptic activity may play a permissive role in the development of glutamatergic synapses. To study the direct roles of NRG2 in the development of glutamatergic synapses, depletion of NRG2 should start after the critical period in order not to interfere with the GABAergic synapse formation. To this end, we started to treat the slice cultures with Dox at 7 dpi (referred to as late Dox treatment), which had no significant effect on GPSCs (Fig. 3 A3, A4).Figure 4 Ashows input–output curves for mean amplitudes of AMPA-EPSCs and NMDA-EPSCs evoked by stimulation of IML at 12 and 15 dpi of newborn GCs grown in control or Dox-treated slices. At 12 dpi, neither AMPA-EPSC nor NMDA-EPSC amplitudes were significantly affected by NRG2 knock-down (Fig. 4 A1, AMPA-EPSC, F (1,16)= 2.17, p= 0.160;Fig. 4 A2, NMDA-EPSC, F (1,17)= 2.33, p= 0.145, RM one-way ANOVA). However, at 15 dpi, both amplitudes were significantly lower in the Dox-treated newborn GCs (Fig. 4 A1, AMPA-EPSC, F (1,16)= 15.2, p< 0.001;Fig. 4 A2, NMDA-EPSC, F (1,17)= 6.78, p= 0.019, RM one-way ANOVA). The late Dox treatment completely suppressed the developmental increase in AMPA-EPSCs and partially suppressed the NMDA-EPSCs to ∼40% of the control (Fig. 4 A, C1). These findings are summarized inFigure 4 B, which displays developmental changes of the mean amplitudes for AMPA-EPSCs and NMDA-EPSCs evoked by 10 V stimulation at control and Dox-treated newborn GCs. The differential effects of NRG2 knock-down on the AMPA-EPSCs and NMDA-EPSCs are more clearly demonstrated by comparing the AMPA/NMDA ratio of control and Dox-treated newborn GCs. AMPA/NMDA ratio was estimated from AMPA-EPSCs and NMDA-EPSCs that were measured in the same cell and evoked by the same stimulation intensity (10 V). Developmental increase of the AMPA/NMDA ratio in newborn GCs was abolished by knock-down of NRG2 (Fig. 4 C2). Because nascent glutamatergic synapses express only NMDA receptors and then AMPA receptors are added as they are matured, the AMPA/NMDA ratio is an index for maturity of glutamatergic synapses (Wu et al., 1996;Akerman and Cline, 2006;Haas et al., 2006). These results indicate that NRG2 is essential for the maturation of glutamatergic synapses in newborn GCs. Open in new tab Download powerpoint Figure 4. NRG2 is essential for maturation of glutamatergic synapses. A1, A2, Averaged input–output curves of AMPA-EPSCs ( A1) and NMDA-EPSCs ( A2) in control (black) and NRG2-depleted (red, Dox-treated from 7 dpi) newborn GCs at 12 dpi (open circles, dotted lines) or at 15 dpi (solid circles, solid lines). Amplitudes of both AMPA-EPSCs and NMDA-EPSCs were significantly lower in Dox-treated GCs at 15 dpi but not at 12 dpi. B, Developmental increases of AMPA-EPSCs ( B1) and NMDA-EPSCs ( B2) in control and NRG2-depleted (Dox-treated from 7 dpi) newborn GCs. Mean amplitudes of EPSCs evoked by 10 V stimulation of IML were plotted as a function of dpi. C1, Representative traces of AMPA-EPSCs and NMDA-EPSCs under control (left) and Dox-treated conditions (right) at 15 dpi. C2, Ratio of AMPA-EPSC to NMDA-EPSC amplitude in control and NRG2-depleted (Dox-treated from 7 dpi) newborn GCs. To test whether NRG2 knock-down has any effect on dendritic growth, we compared the morphology of newborn GCs in hippocampal slices at different dpi's under control or late Dox treatment conditions (Fig. 5 A). The complexity of dendritic arbor was quantified using Sholl analysis (see Materials and Methods for details). The complexity and the total length of dendritic arbors in the Dox-treated GCs were significantly lower than the control GCs, not only at 14 dpi, but also at 10 dpi (Fig. 5 B, Scholl analysis, p< 0.001 at both dpi's, RM one-way ANOVA;Fig. 5 C, cumulative distributions of total dendritic length, p< 0.01, K-S test). The developmental increase of mean values for total dendritic length was significantly retarded in the Dox-treated GCs (Fig. 5 D). Considering that NMDA-EPSCs in the Dox-treated GCs were not significantly different from the control GCs until 13 dpi (Fig. 4 B2), the effect of Dox on the dendritic growth emerged 4 d earlier than that on EPSCs. Because the Dox-induced impairment was specific to dendritic development between 10 and 13 dpi, NRG2 may have a role in the morphological development rather than glutamatergic synaptogenesis during this period. However, it is not clear whether the lack of developmental increase in the AMPA/NMDA ratio is caused by NRG2 depletion itself or secondary to impaired dendritic arborization (see Discussion). Figure 5. Impairment in dendritic growth precedes that in EPSCs in NRG2-depleted GCs. A, Representative dendritic arbors reconstructed from confocal images of control and Dox-treated newborn GCs at 10 and14 dpi. Scale bar, 50 μm. B, Sholl analysis of dendritic complexity at 10 and 14 dpi showing the intersection numbers per 10 μm radial unit distance. The complexity was significantly reduced by NRG2 depletion at both 10 and 14 dpi (control vs Dox, F (1,35)= 19.1 at 10 dpi, F (1,67)= 42.5 at 14 dpi, p< 0.001 for both dpi's, RM one-way ANOVA). C, Cumulative distributions of dendrites at 10 and 14 dpi. p< 0.01; * p< 0,001 by K-S test. D, Developmental increases of total dendritic lengths in control and Dox-treated GCs. *** p< 0.001 ( ttest). GABAergic synapse formation of newborn GCs is mediated by ErbB4 tyrosine kinase Neuregulins are known as ligands of ErbB family of receptor tyrosine kinases (Iwakura and Nawa, 2013). Although NRG2 can bind to both ErbB3 and ErbB4, the NRG2-induced tyrosine kinase activity may be mediated by ErbB4 because ErbB3 lacks kinase activity. To test the involvement of ErbB4 in the downstream signaling of NRG2, we investigated the effect of AG-1478 (10 μ m), an ErbB4 tyrosine kinase inhibitor, on postsynaptic currents in newborn GCs. The amplitudes of feedforward GPSCs recorded in newborn GCs at 7 dpi was significantly lower in the slice culture treated with AG-1478 from 4 dpi (Fig. 6 A1, control vs AG-1478-treated, p< 0.001). However, treatment with AG-1478 from 10 dpi had little effect on the amplitudes of feedforward GPSCs measured in newborn GCs at 14 dpi (Fig. 6 A2, control vs AG-1478-treated, p= 0.878). These results suggest that activation of ErbB4 is required for the developmental increase in GPSCs during the critical period, but not for the later period. Such age dependence of the AG-1478 effect is similar to that of the NRG2 knock-down effects on GPSCs (Fig. 3), suggesting that NRG2 and ErbB4 are on the same signaling pathway required for GABAergic synapse formation. Figure 6. Effects of ErbB4 inhibition on GPSCs and EPSCs. A, B, Effects of ErbB4 inhibitors on GPSCs of newborn GCs. A1, A2, Mean amplitudes of feedforward GPSCs in control (black), Dox-treated (red), and AG-1478-treated (10 μ m, green, denoted as “AG”) newborn GCs. Dox and AG-1478 were added in culture media from 4 dpi ( A1) or 10 dpi ( A2) and feedforward GPSCs were recorded at 7 dpi ( A1) or 14 dpi ( A2), respectively. B, Mean amplitudes of monosynaptic GPSCs at 14 dpi in newborn GCs grown under control conditions or under the condition in the presence of Dox, AG-1478, or anti-ErbB4 antibody (10 μg/ml, purple, denoted as “Ab-3”). Note that the three kinds of treatment reduced the GPSCs to a similar extent when the starting date of treatment was 4 dpi. C, D, Effects of AG-1478 on EPSCs in newborn GCs. Mean amplitudes ( C1, D1) and PPR ( C2, D2) of AMPA-EPSCs ( C) and those of NMDA-EPSCs ( D) in control, Dox-treated, or AG-1478-treated newborn GCs. Dox and AG-1478 were added in culture media from 7 dpi and EPSCs were recorded at 14 dpi. Tx., Treatment. It is possible that AG-1478 may have an effect on glutamatergic synapses on GABAergic interneurons, which in turn may suppress the feedforward GPSCs in newborn GCs. To rule out this possibility, we recorded the monosynaptic GPSCs in AG-1478-treated or control newborn GCs. Consistent with the results of feedforward GPSCs, the amplitudes of monosynaptic GPSCs were lower in newborn GCs treated with AG-1478 from 4 dpi (Fig. 6 B, control vs AG-1478-treated, p< 0.01) and were not affected by AG-1478 treatment from 10 dpi (Fig. 6 B, p= 0.182), arguing against the possible effects of AG-1478 on glutamatergic synapses onto interneurons. The inhibition effect of AG-1478 was significantly larger than the knock-down effect of NRG2 on feedforward GPSCs (Fig. 6 A1, Dox-treated vs AG-1478-treated, p= 0.034). The reason for this might be either that the efficiency of NRG2 knock-down is <100% or that the drug effect is immediate whereas the knock-down of preexisting NRG2 takes effect later. To confirm the involvement of ErbB4, we tested the effect of monoclonal antibody targeting ectodomain of ErbB4 (C-erbB4 Ab-3 antibody, denoted as “Ab-3”). The ErbB4-blocking effect of Ab-3 antibody has been tested previously (Min et al., 2011). Treatment of the slice culture with Ab-3 antibody (10 μg/ml) from 4 dpi suppressed monosynaptic GPSCs measured at 14 dpi to a similar extent as AG-1478, confirming the involvement of ErbB4 in GABAergic synaptogenesis. The PPR was not altered by any of NRG2 depletion or AG-1478 or Ab-3 treatments from 4 dpi ( p> 0.05 for all three conditions), implying that the decrease of monosynaptic GPSC amplitude by NRG2 knock-down or by ErbB4 inhibition is not caused by a change in presynaptic release probability. Because NRG2 plays an essential role in the development of, not only GABAergic, but also glutamatergic synapses in newborn GCs, we tested whether ErbB4 mediates the downstream signaling of NRG2 involved in the latter. Unlike GPSCs, neither AMPA-EPSCs nor NMDA-EPSCs recorded in newborn GCs at 14 dpi were affected by AG-1478 treatment from 7 dpi (control vs AG-1478-treated;Fig. 6 C1, AMPA-EPSCs, p= 0.302;Fig. 6 D1, NMDA-EPSCs, p= 0.942). The PPRs of AMPA-EPSCs and NMDA-EPSCs were not altered by either Dox- or AG-1478 treatment from 7 dpi (Fig. 6 C2, D2). These results suggest that the role of NRG2 in glutamatergic synapse development in newborn GC is independent of ErbB4 tyrosine kinase. NRG2 signaling for glutamatergic synapse maturation and dendritic growth is mediated by its intracellular domain Given that ErbB4 kinase activity is not relevant to the NRG2-dependent development of glutamatergic synapses in newborn GCs, we hypothesized that a reverse signaling pathway of NRG2 is involved. The role of reverse signaling of NRG1 has been studied at glutamatergic synapses of central neurons for the last decade (Bao et al., 2003;Bao et al., 2004;Chen et al., 2010;Fazzari et al., 2014) and involves activity-dependent cleavage and nuclear translocation of intracellular domain of NRG1 (NRG1-ICD). To investigate the possible involvement of NRG2-ICD in glutamatergic synapse development, we designed a retroviral vector expressing myc-tagged NRG2-ICD and TagRFP under the control of Tet-Onand LTR promoters, respectively (Fig. 7 A1). Newborn GCs infected by retrovirus encoding NRG2-ICD could be distinguished from those infected by retrovirus encoding shNRG2mir by the red fluorescence of TagRFP. Using immunohistochemical detection of myc, we confirmed that the newborn GCs infected by the retrovirus reliably express myc-tagged NRG2-ICD in the presence of Dox (Fig. 7 A2). Download figure Figure 7. Effects of NRG2 depletion on glutamatergic synaptic transmission and dendritic arborization are rescued by NRG2-ICD overexpression. A, Retrovirus for overexpression of intracellular domain of NRG2 (NRG2-ICD) in newborn GCs. A1, Schematic of the retroviral cassette for tetracycline-inducible expression of NRG2-ICD in parallel with constitutive expression of TagRFP. A2, Only under the Dox-treated conditions (Dox Tx.) did the TagRFP-positive newborn GCs express c-myc-tagged NRG2-ICD. NRG2-ICD was immunostained with anti-myc IgG. Scale bar, 10 μm. A3, Fluorescence image of a hippocampal slice culture infected with two kinds of retroviruses: shNRG2mir virus and NRG2-ICD virus. Newborn GCs infected with one of the two viruses express EGFP (green) or TagRFP (magenta). Open arrowheads indicate the newborn GCs infected with both viruses simultaneously. Scale bar, 100 μm. A4, Notations for experimental conditions of virus types and Dox treatment. B, C, Concurrent overexpression of NRG2-ICD in the NRG2-depeleted newborn GCs rescued the knock-down effects of shNRG2mir on AMPA-EPSCs ( B1), NMDA-EPSCs ( B2), and the AMPA/NMDA ratio ( B3), but not GPSCs ( C). The amplitudes of AMPA-EPSCs or NMDA-EPSCs of newborn GCs overexpressing only NRG2-ICD were not significantly different ( B1– B3, gray bars) from the control GCs. Dox was applied from 7 to 14 dpi ( B1– B3) or from 4 to 9 dpi ( C). D, Overexpression of NRG2-ICD rescued the impaired dendritic arborization caused by NRG2 knock-down (control, n= 13; NRG K/D, n= 27; rescue, n= 10). D1, Representative morphology of control, NRG2 K/D, and rescued newborn GCs at 14 dpi. Scale bar, 50 μm. D2, Dendritic arbor complexity evaluated by Sholl analysis was not different between control and rescued GCs (control vs rescued, F (1,21)= 2.45, p= 0.774 by RM one-way ANOVA). D3, D4, Cumulative distribution ( D3) and averages ( D4) of total length of dendrites were rescued by NRG2-ICD to the control level (control vs rescue; D3, p= 0.943, K-S test; D4, p= 0.476). Results for control and NRG2-depleted newborn GCs were reproduced from those inFigure 5, B–D. Next, we tested whether the NRG2 knock-down effects on glutamatergic synaptic transmission and dendritic arborization could be rescued by the expression of NRG2-ICD. Hippocampal slice cultures were simultaneously infected with two kinds of retroviruses: retroviruses encoding shNRG2mir plus EGFP (shNRG2mir virus) or NRG2-ICD plus TagRFP (NRG2-ICD virus). Because shNRG2mir targets a different domain of NRG2 other than its ICD, it is expected that endogenous NRG2 is depleted by shNRG2mir, whereas shNRG2mir-resistant NRG2-ICD is overexpressed in the newborn GCs labeled with both EGFP and TagRFP (Fig. 7 A3). We compared postsynaptic currents in newborn GCs under four different conditions as defined inFigure 7 A4. Dox treatment started at 7 and 4 dpi to observe EPSCs and GPSCs, respectively. We found that neither the amplitudes of AMPA-EPSCs and NMDA-EPSCs nor the AMPA/NMDA ratio was significantly different in the doubly labeled newborn GCs (denoted as “rescue” inFig. 7) compared with the control GCs, indicating that the effects of NRG2 depletion is rescued to the control level by the NRG2-ICD overexpression (Figs. 7 B1–B3, control vs rescue, p> 0.1). Conversely, the impaired development of GPSCs caused by NRG2 knock-down was not rescued by NRG2-ICD overexpression (Fig. 7 C, NRG2 K/D vs rescue, p= 0.697). These results demonstrate that reverse signaling of NRG2, mediated by NRG2-ICD, is involved in the development of glutamatergic synapses, but not in GABAergic synapse formation. Finally, we observed the dendritic morphology of EGFP-positive and TagRFP-positive newborn GCs treated with Dox from 7 dpi. Overexpression of NRG2-ICD rescued the impairments of dendritic arborization observed in NRG2-depleted newborn GCs at 14 dpi (Fig. 7 D1). Both the complexity of dendritic arbor and the total dendritic length were rescued to the control level by NRG2-ICD overexpression (Fig. 7 D2, Scholl analysis, F (1,31)= 0.074, p= 0.788, RM one-way ANOVA;Fig. 7 D3, cumulative distributions of total dendritic lengths, p= 0.991, K-S test;Fig. 7 D4, mean total dendritic length, p< 0.001, ttest). These results suggest that reverse signaling of NRG2 is involved in the maturation of glutamatergic synapses and dendritic growth in newborn GCs, whereas forward signaling of NRG2 contributes to GABAergic synaptogenesis. Discussion The present study indicates that NRG2 plays a key role in formation of GABAergic synapses and maturation of glutamatergic synapses in newborn GCs via distinct signaling pathways: forward and reverse signaling of NRG2, respectively. NRG2 expression is very low at birth and notably increases during PW1 in neocortex, hippocampus, and cerebellum with development and decreases by PW3 in most brain regions except dentate gyrus, olfactory bulb, and cerebellum (Longart et al., 2004). Given that GABAergic synaptogenesis starts during PW2 in newborn GCs (Fig. 2 F), it may coincide with the period when NRG2 expression is fully matured. Because the expression of NRG2 is confined to somatodendritic compartments (Longart et al., 2004), NRG2 can act as a ligand for presynaptic ErbB4. Recent immunochemical studies revealed that ErbB4 is expressed on parvalbumin (PV) or cholecystokinin-positive basket cells and neuronal nitric oxide synthase (nNOS)-positive interneurons (Neddens and Buonanno, 2010). Neurogliaform interneurons are a major group of nNOS-positive interneurons (Armstrong et al., 2012). Interestingly, it has been shown recently that newborn GCs receive synaptic inputs from PV-positive and neurogliaform interneurons (Markwardt et al., 2011;Song et al., 2013). It has been suggested that presynaptic terminals of GABAergic interneurons innervating perisomatic regions of principal neurons express ErbB4 and that NRG1-dependent activation of ErbB4 promotes GABAergic synaptogenesis in cortical neurons (Woo et al., 2007;Fazzari et al., 2010; but seeVullhorst et al., 2009). Consistent with these notions, we found that GABAergic synaptogenesis in newborn GCs is suppressed, not only by NRG2 depletion, but also by inhibition of ErbB4. These findings raise the possibility that ErbB4 localized at GABAergic presynaptic terminals, probably of basket cells and/or neurogliaform interneurons, may be responsible for the NRG2-dependent GABAergic synaptogenesis in newborn GCs. Because the expression of ErbB4 in newborn GCs is very weak, it is unlikely that postsynaptic NRG2 on newborn GCs act as an autocrine ligand for postsynaptic ErbB4 even if its ectodomain is cleaved. In contrast to our finding that NRG2 is dispensable for the maintenance of GABAergic synapses in newborn GCs, recent studies showed that the NRG1/ErbB4 interaction enhances GABAergic transmission in neocortex and amygdala (Woo et al., 2007;Lu et al., 2014). This discrepancy may be attributed to the molecular difference between NRG1 and NRG2, to the differences in developmental stages, and/or to the regional difference in the basal interaction level between ErbB4 and neuregulins. There are controversial studies on the role of ErbB4 in glutamatergic synapse maturation. The lack of AG-1478 effects on the development of glutamatergic synapses suggests that the tyrosine kinase activity of ErbB4 is not involved in glutamatergic synapse maturation (Fig. 6 C, D). It has been suggested, however, that presynaptic NRG1-induced activation of postsynaptic ErbB4 enhances AMPA-EPSCs in a kinase-dependent manner (Li et al., 2007) and that ErbB2/4 is involved in the maturation of dendritic spines (Barros et al., 2009). This idea on the role of ErbB4 in glutamatergic synapse maturation was challenged by a more recent study showing little role of ErbB4 expressed on pyramidal neurons in excitatory synaptic transmission (Fazzari et al., 2010). Therefore, the present study is rather consistent with the latter view. Nevertheless, our results do not rule out the involvement of ErbB4 in glutamatergic synapse maturation because interaction of NRG2 with the ectodomain of ErbB4 may trigger the reverse signaling of NRG2 independently of the ErbB4 kinase activity (Bao et al., 2003;Chen et al., 2010). Reverse signaling of neuregulins We showed that the NRG2 knock-down effects on the glutamatergic synapse maturation and dendritic arborization are rescued by the expression of NRG2-ICD (Fig. 7). Furthermore, the development of glutamatergic synapses was independent of ErbB4 tyrosine kinase activity (Fig. 6 C, D). These results suggest that maturation of glutamatergic synapse is mediated, not by forward signaling, but rather by reverse signaling of NRG2. Although reverse signaling of NRG2 has not been studied previously, there is a line of evidence for the reverse signaling of NRG1 type III (Bao et al., 2003;Bao et al., 2004;Chen et al., 2010;Fazzari et al., 2014). The reverse signaling of NRG1 type III requires sequential cleavage of ectodomain and transmembrane domain of NRG1 by TNF-α converting enzyme (TACE) and γ-secretase containing the subunit Aph1b, respectively (Bao et al., 2003;La Marca et al., 2011;Fazzari et al., 2014). The cleaved NRG1-ICDs are translocated into the nucleus and enhance transcription of PSD-95 by interaction with a zinc-finger transcription factor, Eos (Bao et al., 2004). Therefore, any genetic mutations in γ-secretase-dependent intramembranous cleavage site of NRG1 or Aph1b resulted in impairments of dendritic arborization, dendritic spine formation, and glutamatergic synaptic transmission (Chen et al., 2010;Fazzari et al., 2014). Examining the amino acid sequences of NRG2, we found that the γ-secretase and TACE-dependent cleavage sites, as well as the nuclear localization signal, are conserved in NRG2. Because the key sequences are not different between the α and β isoforms of NRG2, both isoforms may mediate reverse signaling (Hobbs et al., 2002). In light of knowledge from NRG1 studies, there is a possibility that NRG2-ICD may function as a transcription regulator, but the identity of transcription factors interacting with NRG2-ICD remains to be elucidated. The expression and cleavage of NRG1 are regulated in an activity-dependent manner (Eilam et al., 1998;Bao et al., 2003;Bao et al., 2004;Ozaki et al., 2004), implying that GABAergic input-induced Ca 2+signaling may be one of factors prerequisite for the reverse signaling of NRG2 in newborn GCs. Previous studies suggested that excitatory GABAergic inputs and subsequent Ca 2+signaling play a key role in coupling the neural activity and the structural plasticity of newborn GCs (Tozuka et al., 2005;Ge et al., 2006;Lee et al., 2012a). Therefore, it is possible that the nurturing effects of network activity on dendritic arborization in newborn GCs may require the reverse signaling of NRG2 as a downstream signaling pathway of the depolarizing GABAergic inputs. Role of NRG2 in dendritic growth and maturation of glutamatergic synapses Given that only NMDA receptors are expressed in nascent glutamatergic synapses, the amplitude of NMDA-EPSCs could be a more appropriate parameter for glutamatergic synaptogenesis than AMAP-EPSCs. The suppression of developmental increase in NMDA-EPSCs can be caused by direct inhibition of glutamatergic synaptogenesis or secondary to the inhibition of dendritic arborization. The present study showed that depletion of NRG2 impairs dendritic development, with little effect on NMDA-EPSCs between 10 and 13 dpi (Figs. 4 B2,5). Therefore, it is unlikely that NRG2 contributes to glutamatergic synaptogenesis. In addition, the impoverished dendritic arbor caused by NRG2 depletion may be responsible for the lower developmental increase of EPSCs that became evident at 14 dpi in Dox-treated GCs. It remains to be elucidated, however, whether maturation of glutamatergic synapses, manifested as an increase in the AMPA/NMDA ratio, is mediated directly by NRG2 or if it is secondary to dendritic growth. Because the effects of NRG2 depletion on the NMDA-EPSCs emerged at the dpi, when AMPA-EPSCs started to increase and its effect on AMPA-EPSCs was more substantial, it is likely that NRG2 contributes directly to maturation of glutamatergic synapses. Differential evaluation of dendritic growth and maturation of glutamatergic synapses in immature GCs may not be easy, especially if dendritic arborization in these cells follows the synaptotrophic hypothesis, which argues that addition of AMPA receptors to a nascent glutamatergic synapse is required for stabilization, not only of the synapse, but also of the associated dendritic branch (Haas et al., 2006;Cline and Haas, 2008). Investigation of NRG2 knock-down effects on EPSCs in mature GCs may help to address this question. Maturation of newborn GCs in hippocampal slice cultures In the present study, we investigated the role of NRG2 by comparing developmental increases in synaptic currents in newborn GCs labeled in organotypic slice cultures taken from the early postnatal rat hippocampus. Two factors should be considered for evaluating the ex vivodata. First, maturation of newborn GCs is known to be faster at a younger age (Overstreet-Wadiche et al., 2006a). As noted in the present study and our previous study (Lee et al., 2012a), GABAergic synapse formation in newborn GCs in the present study was 3 d earlier than that of newborn GCs in the adult hippocampus (Espósito et al., 2005). Second, the development of newborn GCs is affected by abnormal patterns of neural activity of the slice culture. It has been shown that rewiring subsequent to slicing-induced massive deafferentiation leads to epileptiform discharge in the CA3 region (Dyhrfjeld-Johnsen et al., 2010). Given that seizure accelerates the maturation of newborn GCs (Overstreet-Wadiche et al., 2006b), the time course of developmental increases of synaptic current and morphological maturation may be faster than in vivo. Nevertheless, maturation of newborn GCs in the organotypic slice culture was comparable to that in age-matched in vivoconditions (Namba et al., 2007). Indeed, we found that the amplitude of monosynaptic GPSCs reached a plateau 1 or 2 d earlier in the slice culture than newborn GCs labeled in vivoof P16–P22 rats (data not shown). This small difference may be related to the delayed emergence of epileptiform discharge in the culture. It has been shown that epileptiform discharges were first observed at 21 dpi in 50% of the cultures, indicating that our observation window is earlier than the development of epileptiform discharge. The present study was done in the neonatal or juvenile dentate gyrus, but adult neurogenesis recapitulates embryonic development (Espósito et al., 2005). Furthermore, the expression of NRG2 persists in the mature GCs. Therefore, it remains to be elucidated whether NRG2 may play a role in synaptogenesis and/or synaptic plasticity in mature GCs and adult-born GCs. Footnotes This work was supported by the National Research Foundation funded by the Korean Ministry of Science, ICT and Future Planning (Grants 2012-0004904 and 2012-0009135 to S.-H.L. and Grant NRF-2015M3C7A1028790 to W.S.). K.-H.L. is a postdoctorate and C.H.Y. is a graduate student supported by BK21plus. The authors declare no competing financial interests. Correspondence should be addressed to Dr. Suk-Ho Lee, Department of Physiology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea. leesukho@snu.ac.kr References ↵ Abe Y , Namba H , Kato T , Iwakura Y , Nawa H ( 2011 ) Neuregulin-1 signals from the periphery regulate AMPA receptor sensitivity and expression in GABAergic interneurons in developing neocortex . 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Tozuka Y , Fukuda S , Keywords dentate gyrus ErbB4 neuregulin neurogenesis synaptogenesis	https://www.jneurosci.org/content/35/50/16479
Paras Ram v. Kamlesh . \| Punjab & Haryana High Court \| Judgment \| Law \| CaseMine Get free access to the complete judgment in Paras Ram v. Kamlesh . on CaseMine. Paras Ram v. Kamlesh . Punjab & Haryana High Court Sep 24, 1981 Subsequent References CaseIQ TM (AI Recommendations) Paras Ram v. Kamlesh . Case Information CITATION CODES citation codes CASE NO. F.A.F.O No. 70-M of 1980 ATTORNEY(S) JUDGES S.S Sandhawalia, C.J S.P Goyal, J. ACTS Sec. 13(1)(i-a) of the Hindu Marriage Act Paras Ram v. Kamlesh . Page: 61S.S Sandhawalia, C.J:— Would a mere allegation of adultery by a spouse against the other made in defence in a written statement, bv itself amount to cruelty, within the meaning ofSec. 13(1)(i-a) of the Hindu Marriage Actis the core question which has necessitated this reference. 2. Because of reasons which appear hereinafter necessitating a remand to the trial court, it suffices to advert briefly to the facts relevant to the issue aforesaid only. The appellant-husband had preferred the petition for the dissolution of marriage against his wife u/s. 13 of the H.M Act inter alia on the ground of cruelty and desertion. The respondent-wife filed a written statement and subsequently an amendment thereof. Therein whilst denying the factual averments made by the appellant-husband she herself alleged that he had illicit relations with some women in the village and wanted her to leave him forever so that he may either carry on such a liason with them or marry afresh. Equally scurrilous allegation made was that the wife of Sardar Singh, the elder brother of the appellant-husband having died he himself as also his relations wished her to cohabit with Sardar Singh or get Sardar Singh married by exercising the influence of her father. On the aforesaid pleadings the omnibus issue framed by the trial Court was in the following terms:— 1. Whether the petitioner is entitled for dissolution of marriage by divorce on the grounds stated in Para No. 4 of the petition? 2. Relief. The trial Court found the material issue No. 1 against the appellant-husband on a firm finding that neither the ground of desertion nor that of cruelty as alleged in the petition stood proved. It was, however, urged before it that the respondent-wife had made a wild allegation of adultery in her written statement which would per se amount to legal cruelty and, therefore, the appellant-husband was entitled to succeed on that score alone. This stand was rejected on the ground that it was not proved that these wild allegations against the appellant had been made prior to the filing of the petition and having been made merely by way of defence in the written statement these cannot amount to cruelty. As a necessary consequence the petition was dismissed. 3. On appeal when the matter came up before my learned brother S.P Goyal, J., the aforesaid contention that the mere allegation of adultery made in the written statement would amount to mental or legal cruelty was strenuously pressed primarily on the basis ofSmt. Jiwan Lata v. Krishan Kumar, 1979 Cur LJ (Civ) 509. Finding that the observations in Jiwan Lata's case did tend to support the appellant's claim and expressing a doubt about the correctness of its ratio, the matter was referred to a larger Bench. 4. As before the learned Single Judge so before us, the learned counsel for the appellant did not challenge the findings of the trial Court on the facts constituting the pleas of cruelty and desertion already taken in the petition which are accordingly affirmed. For the contention that the scurrilous allegation of adultery levelled by the respondent-wife in her statement would, without more amount to legal cruelty and the appellant was, therefore, entitled to succeed on this ground alone irrespective of his failure to establish the case set up by him, the ratio of Smt. Jiwan Lata's case has inevitably been commended for acceptance on behalf of the appellant. 5. Before adverting to the judgment on which basic reliance is sought to be placed the aforesaid argument deserves some consideration on principle as well. To my mind the inherent fallacy which seems to have crept in here stems from the settled legal position that a false allegation of adultery against a spouse amounts to cruelty in the eye of law. However, it would be a far cry to infer therefrom that an allegation of adultery, whether proved or not, would by itself be the mathematical equivalent of legal cruelty. It is not, and in my view cannot possibly be the law, that a factually Page: 62true allegation of adultery whether made otherwise or in defence in a written statement would amount to cruelty. In this field the truth or otherwise of such an allegation is the crux of the matter. Therefore, before an allegation of adultery can be deemed as legal cruelty it must first be proved as factually false. To highlight, it is a blantantly false allegation of adultery which would amount to legal cruelty, and not possibly a true allegation of that nature which cannot give any cause of action to the offending spouse. Once this is so, it would appear to be elementary that any such allegation of adultery must be put to trial and it is only when its falsity or otherwise is determined that any legal consequence can flow therefrom. It would indeed be going too far to hold that a mere allegation by itself and that too made by way of a defence in legal pleadings should become legal cruelty in the eye of law and per se be the ground for divorce. Such a proposition is unwarranted either on the language of the statute or on principle. This apart, holding so might well frustrate a true and open trial of a matrimonial offence of adultery because it might well inhibit a spouse to raise such a defence, even though it may be factually true. 6. One may now examine the correctness of the view in Smt. Jiwan Lata's case, 1979 Cur LJ (Civ) 509 which provides the cornerstone for the argument on behalf of the appellant. Undoubtedly the observations therein would lend support to such a stand. A close perusal of the judgment however, reveals that on this specific point there is hardly any discussion either on principle or by reference to precedent. It appears that the issue was hardly debated before the learned judge and the observations have been made on first impression. These again seemed to be based on a concession by the counsel for the parties on the larger issue that a false allegation of adultery would amount to cruelty without further analyis about the nature and the modus of proof therefor. The crucial distinction between allegation of adultery made prior to the filing of the case or al unde or in collateral proceedings and such an allegation made by way of defence in the written statement seems to have been totally lost sight of. As already noticed there is no manner of doubt that an allegation of adultery made prior to the filing of the petition and put in issue and found to be false would constitute legal cruelty. However, a mere allegation by way of a defence in the written statement irrespective of its falsity or otherwise is not in the same category unless, of course, such an issue is specifically put to trial and the firm finding arrived at whether the allegation was false or substantiated. 7. In arriving at the conclusion which he did the learned single Judge referred to and has obviously placed reliance onMadan Mohan v. Smt. Sarla Kohli,AIR 1967 Punj & Har 397, andSmt. Dassi Dhani Ram v. Dhani Ram,AIR 1969 Punj & Har 25. An analysis of both of them would show that they pertain to allegation of adultery made earlier than the filing of the petition or in collateral proceedings which were at issue between the parties. These cases are no warrant for the authority that an allegation of this nature made in defence in written statement irrespective of its proof or falsity would by itself be legal cruelty on the basis of which a petitioner can succeed. Learned counsel for the appellant was indeed fair enough to concede that both the aforesaid cases are not directly attracted to the point. It was candidly conceded that there was no other authority to the effect that the mere allegation of adultery without more was itself cruelty even though it has neither been put in issue or tried thereafter. 8. The view taken in Smt. Jiwan Lata's case, 1979 Cur LJ (Civ) 509 if endorsed would tend to lead to curious, if not, startling consequences. One can well visualise a situation where such an allegation of adultery is made by the defending spouse in the written statement and neither the petitioner nor the respondent thereafter led any evidence. On the ratio of Smt. Jiwan Lata's case even though the petitioner would have totally failed to establish the case set up by him he would nevertheless be entitled to succeed on the mere ground that an allegation of adultery by the defending spouse had been made. The petition would thus have to succeed even though the petitioner neither leads evidence nor substantiates his case. Such a startling result did in fact ensue in Smt. Jiwan Lata's case despite the finding that the allegation levelled by the appellant-petitioner had not been proved Page: 63yet the petition for divorce was allowed to succeed. Such a result cannot be easily countenanced because it is well settled that a petitioner must succeed or fall on the case set up by him. 9. With the greatest respect it appears to me that on this specific point Smt. Jiwan Lata's case, (1979 Cur LJ (Civ) 509) does not lay down the law correctly and is hereby overruled. 10. In fairness to the learned counsel for the respondent, Mr. Chandra Singh, we must notice that in the alternative he had gone to the extreme length of contending that anything pleaded by way of defence in a written statement by a respondent cannot be made a ground for attack by the petitioner. To put it in legal terminology it was suggested that the averments in the written statement including allegation of adultery would be privileged in the sense that the petitioner cannot take any advantage thereof. To epitomise in slightly picturesque language the stand was that a shield cannot be turned into a sword. 11. I regret my inability to subscribe to this extreme proposition. Even when pressed learned counsel could cite no authority in support of this stand. On principle, it appears to us that countenancing such as extreme position may well work great public mischief. In substance it would imply that the defending spouse could raise the foulest allegation of adultery or other matrimonial offences against the other without any fear of any retribution. 12. The falsity of the aforesaid stand is also manifest when examined from another angle. The law seems to be set tied that an earlier or collateral allegation of adultery, if false, would immediately give a cause of action to the offended spouse. If that be so, a second petition would obviously lie by a spouse against whom false allegation of adultery has been raised in an earlier petition. Indeed this position was not very fairly controverted by the learned counsel for the respondent. If in a second petition the allegation of adultery made in the earlier could be made as a cause of action, one fails to see why such an accusation in the written statement in the same proceeding should be on a different footing. Subscribing to the view canvassed on behalf of the respondent can only tend to lead to a multiplicity of proceedings which it is always the intent of the law to avoid. I am, therefore, unable to subscribe to any abstract theory of a complete privilege to the allegation of adultery made in a written statement. 13. Once the aforesaid conclusion has been arrived at that a mere allegation of adultery without more does not amount to legal cruelty. It is manifest that in order to succeed on this ground the petitioner must establish the falsity of such an allegation. The burden of proof, however, being a negative burden would in the initial stage be a light one. It would, therefore, become necessary that the petitioner in such a situation would have to amend the petition and plead the false allegation of adultery amounting to cruelty as a specific ground for matrimonial relief. It is only when this has been made a ground of attack that the petitioner can possibly take advantage of such an allegation, if proved false. Unless the truth or falsity of such allegation made in the written statement is put to trial in the manner aforesaid and it is established one way or the other no legal consequences can flow therefrom for the purpose of Sec. 13(1)(1a) of the Act. It is, therefore, necessary in such a situation that not only the requisite amendment should be made but a specific and clear issue with regard thereto be framed so that the parties should go to trial thereon with their eyes open. We cannot but view with disfavour the framing of rather omnibus issue in matrimonial matters as appears to be the situation in the solitary issue framed in his case. It is elementary that in order to succeed in a petition the burden of establishing cruelty u/s. 13(1)(ia) is on the spouse who alleges the same. However, in a case of the present nature where such an allegation has been made in defence in the written statement it would be a negative burden which can easily be discharged by merely averring that the allegation of adultery is false. It would then be for the spose alleging the adultery to substantiate the same. We must now pointedly notice, that. Mr Ashok Aggarwal the learned counsel for the appellant has forthrightly prayed that on the aforesaid view of the law which we have taken he should now be allowed to amend the petition and expressly make the false allegation of Page: 64adultery made in the written statement as a ground for claiming divorce. In view of our overruling Smt. Jiwan Lata's case, (1979 Cur LJ (Civ) 509) learned counsel for the appellant seems to be on impeccable ground in contending that having acted in accordance with the law as it then existed, he is now more than entitled to seek an amendment of the aforesaid nature. We would, therefore, remand the case to the trial Court with the direction that the appellant be allowed to amend his petition, a specific issue thereon be framed, and thereafter to proceed to try the same. The appeal is allowed in the aforesaid terms. Appeal allowed.	https://www.casemine.com/judgement/in/56b494f2607dba348f00fbce
Cancers \| Free Full-Text \| P-REX1-Independent, Calcium-Dependent RAC1 Hyperactivation in Prostate Cancer The GTPase Rac1 is a well-established master regulator of cell motility and invasiveness contributing to cancer metastasis. Dysregulation of the Rac1 signaling pathway, resulting in elevated motile and invasive potential, has been reported in multiple cancers. However, there are limited studies on the regulation of Rac1 in prostate cancer. Here, we demonstrate that aggressive androgen-independent prostate cancer cells display marked hyperactivation of Rac1. This hyperactivation is independent of P-Rex1 activity or its direct activators, the PI3K product PIP3 and Gβγ subunits. Furthermore, we demonstrate that the motility and invasiveness of PC3 prostate cancer cells is independent of P-Rex1, supporting the analysis of publicly available datasets indicating no correlation between high P-Rex1 expression and cancer progression in patients. Rac1 hyperactivation was not related to the presence of activating Rac1 mutations and was insensitive to overexpression of a Rac-GAP or the silencing of specific Rac-GEFs expressed in prostate cancer cells. Interestingly, active Rac1 levels in these cells were markedly reduced by elevations in intracellular calcium or by serum stimulation, suggesting the presence of an alternative means of Rac1 regulation in prostate cancer that does not involve previously established paradigms. P-REX1-Independent, Calcium-Dependent RAC1 Hyperactivation in Prostate Cancer by Martin J. Baker 1 , Martín C. Abba 2 , Rafael Garcia-Mata 3 and Marcelo G. Kazanietz 1,* Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, 1256 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA Author to whom correspondence should be addressed. Cancers 2020 , 12 (2), 480; https://doi.org/10.3390/cancers12020480 Received: 21 December 2019 / Revised: 7 February 2020 / Accepted: 17 February 2020 / Published: 19 February 2020 Abstract : The GTPase Rac1 is a well-established master regulator of cell motility and invasiveness contributing to cancer metastasis. Dysregulation of the Rac1 signaling pathway, resulting in elevated motile and invasive potential, has been reported in multiple cancers. However, there are limited studies on the regulation of Rac1 in prostate cancer. Here, we demonstrate that aggressive androgen-independent prostate cancer cells display marked hyperactivation of Rac1. This hyperactivation is independent of P-Rex1 activity or its direct activators, the PI3K product PIP 3 and Gβγ subunits. Furthermore, we demonstrate that the motility and invasiveness of PC3 prostate cancer cells is independent of P-Rex1, supporting the analysis of publicly available datasets indicating no correlation between high P-Rex1 expression and cancer progression in patients. Rac1 hyperactivation was not related to the presence of activating Rac1 mutations and was insensitive to overexpression of a Rac-GAP or the silencing of specific Rac-GEFs expressed in prostate cancer cells. Interestingly, active Rac1 levels in these cells were markedly reduced by elevations in intracellular calcium or by serum stimulation, suggesting the presence of an alternative means of Rac1 regulation in prostate cancer that does not involve previously established paradigms. Keywords: Rac1 ; P-Rex1 ; Rac-GEF ; prostate cancer cells 1. Introduction Prostate cancer is the most frequently diagnosed malignancy in men in the United States aside from skin cancer, with an estimated 175,000 new cases in 2019, and the second leading cause of cancer-related deaths in men [ 1 ]. The 5-year survival rate for patients with localized prostate cancer is >99%. However, this is dramatically reduced to 30% in patients with metastatic prostate cancer [ 1 ]. Aggressive metastatic phenotypes are associated with androgen independence, resulting in resistance to androgen ablation or castration therapies (“Castration-Resistant Prostate Cancer” or CRPC). Indeed, prostate cancer cells that have acquired androgen independence have a significant propensity to metastasize. There is currently no treatment available to cure such androgen-independent prostate cancer, and a greater understanding of the mechanisms that drive CRPC is therefore essential [ 2 ]. The small GTPases Rac1 and Cdc42 have been recognized as major players in cancer progression and perform essential roles in metastasis [ 3 , 4 , 5 ]. These G-proteins are members of the Rho-GTPase family, and as such they are binary switches that cycle between an active GTP-bound state and an inactive GDP-bound state. The activation of these GTPases occurs through the action of Guanine nucleotide Exchange Factors (GEFs), which stimulate the release of GDP and therefore allow the subsequent binding of GTP. Conversely, GTPases are deactivated by the hydrolysis of GTP to GDP, which is accelerated by GTPase Activating Proteins (GAPs) [ 6 ]. A third class of regulatory proteins, called RhoGDIs, also exert influence on the Rho-GTPase activation cycle, in this case by binding the inactive form and sequestering it in the cytosol [ 7 ]. When active, these GTPases and their effectors regulate the generation of actin-rich membrane protrusions that play important roles in cell migration and invasion, with Rac1 driving lamellipodia formation and Cdc42 driving filopodia formation [ 5 , 8 ]. Furthermore, Rac1 has been shown to have additional functions that contribute to cancer progression, including ROS formation [ 9 , 10 ], proliferation [ 4 ], gene expression [ 11 , 12 ] and ribosomal biogenesis [ 13 , 14 ]. The dysregulation of Rac1 activation has been reported in multiple diseases, including cancer [ 3 , 4 , 5 ]. Oncogenic signaling by Rac1 can occur from a mutation, as seen in melanoma with the Rac1 P29S mutant [ 15 , 16 ], or alternatively from dysregulation of the activation cycle. This includes enhanced activation of membrane receptors or their effectors (e.g., PI3K), up-regulation/mutations of Rac-GEFs, or down regulation of Rac-GAPs [ 3 , 6 , 17 , 18 ]. Elevated Rac1 activation has been largely associated with the progression of various cancers and metastatic dissemination of cancer cells. Specifically, for prostate cancer, Rac1 signaling has been implicated in androgen receptor signaling leading to proliferation [ 19 , 20 ] and epithelial-to mesenchymal transition (EMT) [ 21 , 22 ]. Recent work has also demonstrated that the spatial regulation of Rac1 activation in prostate cancer cells, an essential component of directed cell migration, is dependent on RhoH signaling [ 23 ]. The potential involvement of Rac-GEFs in prostate cancer has received little attention. There are two families of Rac-GEFs, the dbl-like GEFs and the DOCK GEFs. One of the few Rac-GEFs characterized in prostate cancer is P-Rex1, a dbl-like exchange factor originally identified in neutrophils and later found to be involved in the progression of specific cancer types such as breast cancer and melanoma [ 24 , 25 , 26 , 27 , 28 ]. P-Rex1 nucleotide exchange activity is dependent on the synergistic stimulation by the PI3K product PIP 3 and Gβγ subunits released from activated heterotrimeric G-proteins [ 24 ]. Consistent with this regulation, P-Rex1 is an effector of tyrosine-kinase receptors (e.g., EGFR, ErbB3 and IGF-IR) and GPCRs (e.g., CXCR4 and FPR1), mediating Rac1-dependent migratory responses in response to their stimulation [ 26 , 29 , 30 ]. Importantly, P-Rex1 overexpression has been shown in various cancer types, particularly luminal breast cancer and prostate cancer [ 26 , 28 ]. It has been reported that P-Rex1 is highly expressed in PC3 prostate cancer cells relative to androgen-dependent cell lines, and that silencing P-Rex1 in PC3 prostate cancer cells leads to a reduction in activated Rac1, as well as in the migratory and invasive capacity of these cells [ 28 ]. Towards the goal of better understanding the relevance of Rac1 signaling in prostate cancer, here, we investigated the activation status and mechanisms of regulation of Rac1 in prostate cancer cell models. We found that activated levels of Rac1 are prominently elevated in the androgen-independent DU145 and PC3 cell lines. An exhaustive analysis of the mechanisms implicated in Rac1 hyperactivation revealed that this dysregulated signal is independent of P-Rex1 activity or other Rac-GEFs expressed in prostate cancer cells. Moreover, we made the unexpected observation that elevated Rac1-GTP levels in androgen-independent prostate cancer cells cannot be reduced by expression of a Rac-GAP, but on the other hand they are sensitive to elevations in intracellular calcium, suggesting alternative regulatory mechanisms for this small GTPase in prostate cancer models. 2. Results 2.1. Aggressive Androgen-Independent Prostate Cancer Cells Display Elevated Rac1-GTP Levels In order to establish whether changes in Rac1 expression or activation occur in aggressive prostate cancer cells, we first determined the total and active (GTP-bound) Rac1 levels. For this analysis, we used non-transformed epithelial cancer cells (RWPE-1, BPH-1), androgen-dependent LNCaP cells, its derivatives C4 and C4-2, as well as aggressive androgen-independent DU145, PC3 and PC3-ML cell lines. Although no major differences in total Rac1 levels could be detected between the prostate cell lines, the assessment of Rac1-GTP levels by a PAK-binding domain (PBD) pull-down assay revealed significantly higher levels of active Rac1 in androgen-independent DU145, PC3, and PC3-ML cells relative to the other cell lines. A quantitative analysis of Rac-GTP levels normalized to total Rac1 showed ~10–15-fold elevation in active Rac1 in DU145, PC3 and PC3-ML cells compared to LNCaP cells ( Figure 1 A,C). A similar trend was observed for the related small GTPase Cdc42, with the highest Cdc42-GTP levels found in PC3 and PC3-ML cells ( Figure 1 A,D). These differences were even larger when Rac1-GTP and Cdc42-GTP levels were determined in serum-starved cells, particularly in PC3 and PC3-ML cells (~25–35-fold higher for Rac1-GTP and 10–15-fold higher for Cdc42-GTP, respectively, relative to LNCaP cells) ( Figure 1 B–D). This was also seen when the androgen-independent PC3 cells were compared to androgen-dependent VCaP prostate cancer cells, both in the presence and absence of FBS ( Figure S1A–D ). Estimation of the active fraction of these GTPases in PC3 cells revealed that 7% of Rac1 and 18% of Cdc42 were active in the presence of 10% FBS. These values were higher in serum-starved cells (15% for Rac1-GTP and 31% for Cdc42-GTP) ( Figure 1 E). Taking into consideration that serum and growth factors activate Rac1 and Cdc42 in most cellular models, the elevated Rac1 and Cdc42 activation after FBS removal was unanticipated. Hyperactivation of Rho GTPases may be the consequence of mutations (e.g., Rac1 P29S in melanoma). To assess whether this was the case in the prostate cancer cells, we carried out Sanger sequencing. This analysis revealed no mutations in Rac1 or Cdc42 in PC3, DU145 or LNCaP cells. Likewise, no mutations were found in RhoGDI1 or RhoGDI2 in these cell lines. This agrees with genome sequence data available from the cancer cell lines encyclopedia (CCLE) ( http://www.broadinstitute.org/ccle ). Western blots also failed to reveal RhoGDI1 and RhoGDI2 loss of expression in the aggressive prostate cancer cell lines that could have explained Rac1 and Cdc42 hyperactivation ( Figure 1 F). No expression of RhoGDI3 could be detected by qPCR in any prostate cell lines used in this study. Similarly, we did not observe any appreciable correlation between the expression of the constitutively active Rac1b variant and Rac1-GTP levels in the prostate cell models ( Figure 1 G). 2.2. Elevated Rac1-GTP in Prostate Cancer Is Independent of P-Rex1 A previous study linked the PI3K- and Gβγ-regulated Rac-specific GEF P-Rex1 to metastatic phenotypes in prostate cancer. The up-regulation of P-Rex1 in PC3 cells relative to LNCaP cells or non-transformed prostate epithelial cells has been associated with Rac1 activation and migration [ 28 ]. We therefore sought to define whether P-Rex1 was responsible for the high basal Rac1-GTP levels observed in prostate cancer cell lines. To address this question, we knocked down P-Rex1 in PC3 cells with a pooled RNAi, which caused >90% reduction in P-Rex1 protein levels. Our results revealed that silencing P-Rex1 did not have any effect on Rac1-GTP levels in PC3 cells ( Figure 2 A). Silencing other PI3K-dependent Rac-GEFs expressed in prostate cancer cells, namely Tiam1, Vav2 or Vav3, also failed to reduce Rac1-GTP levels in PC3 cells ( Figure 2 A and Figure S1G ). To further validate the P-Rex1 independence of Rac1 activation in prostate cancer cells, we examined the effect of the PI3K inhibitor BKM120 and gallein, an agent that disrupts Gβγ signaling. As shown in Figure 2 B,C, neither of these agents were able to reduce Rac1-GTP levels in serum-starved DU145 or PC3 cells. As a control for the inhibitors, we measured the activation of Rac1 by the ErbB3 ligand heregulin-1 (HRG) in MCF-7 breast cancer cells, a response known to be dependent on P-Rex1 activity [ 26 ]. In this case, both BMK120 and gallein significantly reduced HRG-induced Rac1 activation. The efficacy of the PI3K inhibitor was also confirmed by its ability to reduce phospho-Akt levels in PC3 and in HRG-stimulated MCF-7 cells. BKM120 and gallein also failed to reduce Cdc42-GTP levels in DU145 and PC3 cells ( Figure S1E,F ). These results suggest that Rac1 and Cdc42 hyperactivation in androgen-independent prostate cancer cells does not involve aberrant upstream signaling mediated by PI3K/Gβγ -dependent exchange factors, such as P-Rex1. We then sought to establish whether P-Rex1 plays a role in prostate cancer cell migration and invasion. Consistent with the lack of involvement for P-Rex1 in regulating Rac1 activation in PC3 cells, silencing the expression of this Rac-GEF failed to impair cell motility, as determined with a Boyden chamber assay ( Figure 2 D,E). Likewise, the ability of PC3 cells to invade through Matrigel was not affected by P-Rex1 RNAi mediated depletion ( Figure 2 F). Taken together, these results confirmed that P-Rex1 does not contribute to maintaining high Rac1-GTP levels and is dispensable for migration or invasion of androgen-independent prostate cancer cells. Analysis of P-Rex1 mRNA expression showed a prominent up-regulation in androgen-independent prostate cancer cells, most prominently in the bone metastatic PC3-ML variant ( Figure 3 A). However, analysis of publicly available databases did not show appreciable changes of P-Rex1 expression in normal vs. human prostate tumors ( Figure 3 B). In addition, no significant changes in P-Rex1 expression were detected based on Gleason Score ( Figure 3 C). There were also no differences in disease free survival (DFS) based on P-Rex1 mRNA expression ( Figure 3 D). Altogether, this analysis suggests that P-Rex1 may not have a significant role in human prostate cancer progression, unlike that described in other cancer types. 2.3. Elevated Rac1 Activation in Androgen-Independent Prostate Cancer Cells Is Insensitive to the Rac-GAP β2-Chimaerin One explanation for the observed hyperactivation of Rac1 in androgen-independent prostate cancer cells is that it could be the consequence of deficient expression of GAPs responsible for Rac1 inactivation. To address this issue, we overexpressed the Rac-specific GAP β2-chimaerin in prostate cancer cells using an adenoviral approach [ 35 ]. β2-chimaerin was chosen based on its robust ability to deactivate Rac1 in other cell types [ 35 , 36 , 37 ]. To our surprise, ectopic expression of β2-chimaerin in PC3 or DU145 cells did not significantly affect Rac-GTP levels ( Figure 4 A). On the other hand, β2-chimaerin abrogated Rac1 activation by HRG in MCF-7 breast cancer cells, as we have reported in a previous study [ 38 ] ( Figure 4 B). As a control, ectopically expressed β2-chimaerin could be pulled down from both PC3 and MCF-7 cell extracts using a constitutively active Rac1 Q61L bait ( Figure S2A ), as expected based on the preferential ability of Rac-GAPs to bind activated Rac1. Therefore, elevated Rac1-GTP levels in androgen-independent prostate cancer cells are insensitive to Rac-GAP inactivation. The small GTPase RhoA has previously been shown to negatively regulate Rac1 via the activation of a Rac-GAP in other systems [ 39 ]. However, treatment of PC3 cells with the small molecule Rho Activator II [ 40 ] had no effect on the level of Rac1 activation, therefore demonstrating that the pool of hyperactive Rac1 in these prostate cancer cells is also insensitive to negative regulation by RhoA ( Figure S2B ). 2.4. Silencing Rac-GEFs in PC3 Cells Did Not Impact Rac1-GTP Levels The insensitivity of Rac1-GTP levels to Rac-GAP overexpression could be due to the activity of a particularly dominant Rac-GEF driving Rac1 hyperactivation. To address this issue, we decided to use an RNAi approach to individually silence Rac-GEFs expressed in aggressive prostate cancer cells. First, we examined the expression of Rac-GEFs in PC3 cells using qPCR ( Figure S3A ). The analysis showed that 34 GEFs (23 dbl-like GEFs and 11 DOCK GEFs) could be detected at the mRNA level. We were unable to detect the expression of RasGRF1, ARHGEF15, ECT2L, VAV1, MCF2, FGD5, KALRN, RasGRF2 and ARHGEF19. Stable GEF-depleted PC3 cell lines were then generated upon infection with pre-designed shRNA lentiviruses, followed by puromycin selection. For each GEF we used 2–5 different shRNA lentiviruses, totaling 110 puromycin-resistant stable cell lines for a total of 34 GEFs. We then selected for each GEF the stable cell line with the greatest level of silencing, as determined by qPCR ( Figure S3B ), and examined their Rac1-GTP levels using a G-LISA approach. Stable depletion of the GEFs DOCK2, DOCK6 and DOCK11 by any of the viruses used was deemed insufficient (<50%) and these were therefore not included in the analysis. As shown in Figure 5 A, none of the GEF-deficient cell lines showed major reductions in Rac1-GTP levels (normalized to total Rac1) relative to parental cells or cells subject to non-target control shRNA, other than a relatively minor effect for DOCK4-, TIAM2- and ALS2-depleted PC3 cell lines. However, a similar analysis carried out using siRNA duplexes for DOCK4, TIAM2 or ALS2 revealed no change in Rac1-GTP levels ( Figure 5 B and Figure S4A ), suggesting that the effect observed with shRNAs for these GEFs was most likely an off-target effect. Of note, silencing the expression of these GEFs did not cause a reduction in the Cdc42-GTP levels ( Figure S4B ). An increase in Rac-GTP levels in the G-LISA screening was observed upon silencing P-Rex2 and ARHGEF4. However, this effect was most likely a consequence of a reduction in total Rac1 levels that translated into an elevated Rac1-GTP/total Rac1 ratio. Based on these results, we concluded that no single Rac-GEF tested is responsible for Rac1 hyperactivation in prostate cancer. 2.5. Identification of Rac1 Interacting Partners in Prostate Cancer Cells Using Mass Spectrometry In a further effort to elucidate the mechanisms responsible for mediating Rac1 hyperactivation in androgen-independent prostate cancer cells, we performed mass spectrometry with the goal of identifying Rac1 binding partners. For this analysis, we used a GST-tagged G15A Rac1 mutant (nucleotide-free) as a bait, an approach that is used to pull-down associated GEFs [ 41 ]. Mass spectrometry analysis identified 301 potential Rac1 binding partners in PC3 cell lysates ( Table S1 ). Notably, none of these Rac1 associated proteins were known Rac-GEFs, further reinforcing the lack of involvement of these nucleotide-exchange factors. The analysis revealed a number of proteins that based on the literature could be potentially involved in regulating Rac1 signaling. The most prominent candidates were SmgGDS (a Rac1 interacting protein that contributes to its activation by Rac-GEFs [ 42 ]) and Grb2 (shown to facilitate the activation of Rac1 by a GEF [ 43 ]). Therefore, we used a silencing approach to knockdown SmgGDS and Grb2 expression, followed by the determination of Rac-1-GTP levels. These experiments revealed that neither of these interacting proteins were implicated in maintaining elevated Rac1-GTP levels in PC3 cells ( Figure S5A,B ). 2.6. Rac1 Hyperactivation in PC3 Cells Is Reduced in Response to Serum and Elevations in Intracellular Calcium As shown in Figure 1 , subjecting PC3 cells to 24 h serum starvation resulted in an increase in Rac1-GTP and Cdc42-GTP levels relative to cells growing in medium with FBS. Notably, addition of 10% FBS to serum starved PC3 cells led to a rapid decrease in Rac1-GTP and Cdc42-GTP levels. Indeed, 65% and 40% reductions in Rac-GTP and Cdc42-GTP levels, respectively, were observed at 1 min after FBS addition, which progressively returned to the high basal levels after 20–60 min ( Figure 6 A). In contrast, androgen-dependent LNCaP prostate cancer cells, which have a low basal Rac1-GTP level, displayed a moderate increase in Rac1-GTP in response to serum, peaking at 15 min ( Figure 6 B). In an effort to identify the mechanisms responsible for this effect, we examined candidate second messengers capable of affecting Rac1 and Cdc42 activation. Considering that multiple extracellular cues lead to elevations in calcium levels, we examined the effect of the calcium ionophore A23187. Notably, treating PC3 cells with this ionophore led to a rapid and sustained reduction in Rac1-GTP and Cdc42-GTP that was not observed with the vehicle DMSO ( Figure 6 C,D). As a second approach to evaluate the role of intracellular calcium in small GTPase deactivation, we evaluated the effect of thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitor that promotes a significant elevation in intracellular calcium [ 44 ]. Like the calcium ionophore, thapsigargin also reduced Rac1-GTP levels in PC3 cells ( Figure 6 E). In contrast, stimulation of cAMP production with forskolin did not cause a significant effect ( Figure 6 F). A quantitative analysis of these effects is depicted in Figure 6 G. Therefore, this sensitivity to thapsigargin and the ionophore A23187 demonstrates a role for calcium-signaling events in mediating the rapid inactivation of Rac1-GTP and Cdc42-GTP in PC3 cells. 3. Discussion In this study, we investigated the regulation of Rac1 in prostate cancer cellular models. The main findings of our study are three-fold. First, we demonstrated that aggressive androgen-independent prostate cancer cell lines have markedly increased levels of activated Rac1, an effect also observed for the related G-protein Cdc42. Second, we demonstrated that the Rac-GEF P-Rex1, which has been previously reported as a key Rac1 activator in prostate cancer models, is dispensable for conferring Rac1 hyperactivation. Along this line, no other Rac1-GEF or mutations in Rac1 itself or in RhoGDIs could explain the elevation of Rac1-GTP in androgen-independent prostate cancer cells. Third, the elevated Rac1-GTP levels in these cells are not subject to the typical regulatory mechanisms described for Rac GTPases in other contexts, as judged by the unexpected insensitivity to a Rac-GAP and its deactivation via calcium-mediated mechanisms. This raises the possibility that yet unidentified regulatory mechanisms take place in the control of Rac1 activation in prostate cancer cells. Contrarily to a previous report implicating P-Rex1 up-regulation in the maintenance of Rac1 hyperactivation [ 28 ] in androgen-independent prostate cancer cells, our study shows no effect of P-Rex1 silencing on Rac1-GTP levels in this model. Consistent with this, P-Rex1 was also found to be dispensable for the migration or invasion of PC3 cells. Although Qin et al. [ 28 ] reported a reduction in Rac1-GTP levels in PC3 cells by delivery of a single P-Rex1 siRNA duplex. In our hands, the delivery of multiple RNAi duplexes failed to have any effect. The observed differences may relate to the different experimental approaches in each case. In support of our finding, PI3K and Gβγ inhibitors, which abrogate P-Rex1 activation, had no effect on Rac1-GTP levels in androgen-independent prostate cancer cells. It is important to note, however, that the early study [ 28 ] reported significant reductions in CXCR4- and EGFR-mediated PC3 motility, which fits with the proposed mechanisms of P-Rex1 activation by receptor stimulation. In this regard, up-regulated P-Rex1 levels in luminal breast cancer cells do not cause marked changes in basal Rac1-GTP but rather sensitize Rac1 activation to the stimulation of tyrosine-kinase receptors and GPCRs [ 26 , 30 ]. Previous work also identified an up-regulation of P-Rex1 mRNA in PC3 cells compared to less aggressive prostate cancer cells such as LNCaP [ 28 ], similar to our observation in PC3 and PC3-ML cells. However, analysis of publicly available human gene expression databases revealed no correlation of P-Rex1 expression with cancer status, Gleason score or disease-free survival. Therefore, the observed up-regulation of P-Rex1 in PC3 cells does not seem to be representative of a general occurring effect in human prostate cancer. These results argue that P-Rex1 may not represent a promising therapeutic target or biomarker of prostate cancer progression as otherwise suggested in luminal breast cancer and melanoma [ 27 , 30 , 45 ]. Our screening of Rac-GEF-depleted PC3 cell lines was unable to identify a single exchange factor responsible for Rac1 hyperactivation. There are several possible scenarios explaining this result. One is that multiple GEFs are required for maintaining Rac1-GTP and Cdc42-GTP in these cells, and thus silencing single GEFs is insufficient to cause an effect. We cannot rule out that a Rac-GEF not included in our screen could be involved, albeit a less likely possibility because our analysis encompasses the majority of known Rac-GEFs [ 3 ]. However, it is quite remarkable that no Dbl or DOCK family GEFs were identified in our proteomics analysis using a nucleotide free Rac1 bait, which is designed to pull-down Rac1 associated GEFs. Interestingly, the proteomics detected the atypical GEF SmgGDS, a protein reported to interact with Rac1 that indirectly contributes to Rac1 regulation [ 42 , 46 ]; however, this atypical GEF was found to be dispensable for Rac1 hyperactivation in androgen-independent prostate cancer cells. Another possible alternative explanation for the lack of dependency on any of the tested GEFs is that a non-GEF protein promotes or is required for maintaining the active Rac1 and Cdc42 status. For example, a protein could stay bound to the GTPases to prevent their inactivation by GAPs. This would indeed fit with our observation that β2-chimaerin overexpression failed to reduce Rac1-GTP levels in both DU145 and PC3 prostate cancer cells while still achieving a reduction in MCF-7 breast cancer cells. One such candidate is IQGAP1, a scaffold protein that binds to Rac1-GTP and Cdc42-GTP and contributes to the organization of a multimolecular complex involved in regulation of the actin cytoskeleton [ 47 ]. Although IQGAP1 was not identified in our proteomics screen, suppressing its expression by RNAi did not affect Rac1 or Cdc42 hyperactivation in PC3 cells ( Figure S5C ). Another scenario is that the Rac1-GTP pool in androgen-independent prostate cancer cells is located in a cellular compartment that is not accessible to regulatory proteins. In this regard, Rac1 could be localized to distinct organelles where it has specialized functions. However, in the absence of suitable tools to visualize the intracellular localization of endogenous Rac1-GTP we could not address this question. Finally, our results revealed an unexpected mechanism of Rac1 and Cdc42 deactivation in aggressive androgen-independent prostate cancer cells. The observation that PC3 cells display elevated Rac1 and Cdc42 activation in response to serum starvation suggests the presence of a factor in the serum that was suppressing the activation of these GTPases. This was confirmed by the effect of FBS stimulation on starved cells, which produced a rapid reduction in the levels of Rac1-GTP and Cdc42-GTP. This loss of active G-protein could be mimicked by pharmacological agents that increase intracellular calcium levels, either a calcium ionophore or a SERCA pump inhibitor, but not by a cAMP-generating agent. These results were unforeseen since the stimulation of cells with FBS or growth factors is generally expected to increase Rac1 activation, as we observed in LNCaP cells and as previously reported in other cell types [ 48 , 49 ]. Furthermore, calcium-signaling events in other cell types and contexts has been shown to indirectly regulate the activation of Rac1 [ 50 , 51 , 52 ]. It is therefore remarkable that a rise in intracellular calcium results in the deactivation of Rac1 and Cdc42 in PC3 cells and may indicate the presence of a potent calcium-stimulated GAP or the calcium dependent dissociation of a binding partner that otherwise sustains hyperactivation of these GTPases. Such a mechanism may be quite restricted by the localization of Rac1 and Cdc42 in androgen-independent prostate cancer cells. In summary, our work identified Rac1 and Cdc42 hyperactivation in aggressive androgen-insensitive DU145 and PC3 prostate cancer cell lines. These proteins and their regulators are known to play important roles in the progression of cancer and are therefore of interest as potential therapeutic targets [ 3 ]. We have also demonstrated that targeting P-Rex1, a previously identified therapeutic target candidate, is unlikely to have an impact on Rac1 activation status, migration or invasion in prostate cancer cells. Considering the essential roles Rac1 and related GTPases proteins have in metastasis, deciphering the molecular mechanisms behind their unusual regulation in prostate cancer is of utmost importance. 4. Materials and Methods 4.1. Cell Lines, Cell Culture, and Reagents Human prostate cancer cell lines (LNCaP, LNCaP-C4, LNCaP-C4-2, DU145, PC3) and normal prostate epithelial cells (RWPE1) were obtained from ATCC (Rockville, MD, USA). PC3-ML cells were obtained from Dr. Alessandro Fatatis (Drexel University, Philadelphia, PA, USA), as previously described [ 53 ]. BPH-1 cells were a kind gift from Dr. Cecilia Caino (University of Colorado, Denver, CO, USA). VCaP cells were kindly provided by Dr. Trevor M. Penning (University of Pennsylvania, Philadelphia, PA, USA). Heregulin-β1 was purchased from R&D Systems (Minneapolis, MN, USA) and FBS from Hyclone. BKM120 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Gallein was purchased from Tocris Bioscience (Bristol, UK). Forskolin, A123187 and thapsigargin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rho Activator II was purchased from Cytoskeleton (Denver, CO, USA). 4.2. RNA Interference (RNAi) Transfection of siRNA duplexes was performed with Lipofectamine RNAiMax following the manufacturer’s protocol. The following ON-TARGET RNAi duplexes were obtained from Dharmacon (Lafayette, CO, USA): P-Rex1 (L-010063-01, J-010063-10, J-010063-11), Vav2 (L-005199-00), Vav3 (L-010178-00), Als2 (L-014168-00), DOCK4 (L-017968-01), Tiam2 (L-008434-00), SmgGDS (J-015881-05), Grb2 (J-019220-07), IQGAP1 (L-004694-00) and non-target control (LD-001810-10). 4.3. Generation of Stable GEF-Depleted Cell Lines Lentiviral constructs targeting Rac-GEFs and pLKO non-targeting control shRNA constructs were obtained from Sigma-Aldrich. pLKO.1 shRNAs for human ABR (TRCN0000047948), ALS2 (TRCN0000047805), ARHGEF2 (TRCN0000003175), ARHGEF4 (TRCN0000047559), ARHGEF6 (TRCN0000007403), ARHGEF7 (TRCN0000047596), ARHGEF16 (TRCN0000047505), ARHGEF18 (TRCN0000047534), ARHGEF19 (TRCN0000048239), ARHGEF38 (TRCN0000106214), BCR (TRCN0000000789), DOCK1 (TRCN0000029077), DOCK3 (TRCN0000122974), DOCK4 (TRCN0000039730), DOCK5 (TRCN0000113802), DOCK7 (TRCN0000129042), DOCK8 TRCN0000122957), DOCK9 (TRCN0000141047), DOCK10 (TRCN0000122950), ECT2 (TRCN0000047684), FARP2 (TRCN0000047259), FGD5 (TRCN0000048244), FGD6 (TRCN0000047268), KALRN (TRCN0000001430), NGEF (TRCN0000047657), PLEKHG3 (TRCN0000048681), PLEKHG4B (TRCN0000156705), PREX2 (TRCN0000157338), SOS1 (TRCN0000048143), SOS2 (TRCN0000048153), TIAM2 (TRCN0000107211), TRIO (TRCN0000000871), and VAV3 (TRCN0000047699) were obtained from Open Biosystems (Huntsville, AL, USA). Lentiviruses were produced by the Lenti-shRNA Core Facility (UNC-Chapel Hill, NC, USA). Cells were infected with lentivirus particles overnight before selection the following day with puromycin (1 μg/mL). 4.4. Rac1-GTP/Cdc42-GTP Pull-Down (PBD) Assays The pull-down assay for determining Rac1-GTP and Cdc42-GTP levels was performed as previously described and is based on the binding of the active G-protein to the PAK-binding domain (PBD) [ 36 , 54 ]. For some experiments, cells were serum starved for 24 h before the assay. 4.5. G-LISA Assay for Rac1-GTP The colorimetric G-LISA kit (BK128, Cytoskeleton, Denver, CO, USA) was used to measure Rac1-GTP levels in the stable GEF-depleted cell lines. Briefly, cells were grown for 24 h, followed by 24 h serum starvation immediately prior to the assay. Cells were lysed with kit lysis buffer, lysates clarified and then normalized for total protein. G-LISA was performed following the manufacturer’s protocol. The G-LISA signal was normalized for total Rac1 levels that were assessed by Western blot. Experiments were done in triplicate. 4.6. Adenoviral Expression of β2-Chimaerin Ectopic expression of β2-chimaerin was achieved with an adenoviral approach, as previously described [ 37 , 38 ]. The stimulation of cells, when required, was achieved with the addition of 10× stimulant to the cells in starvation media. To simultaneously analyze the activation of Rac1 and Cdc42, post-clarification lysates were divided onto separate beads for the pulldown. 4.7. Western Blot Analysis Western blot was performed as previously described [ 55 ]. The following antibodies were used at 1:1000 dilution: anti-Rac1 clone 23A8 (Cat. # 05-389), anti-Rac1b (Cat. # 09-271) and anti-P-Rex1 (Cat. #MABC178) from EMD Millipore (Burlington, MA, USA); anti-Cdc42 clone 11A11 (Cat. # 2466), anti-phospho-AKT (Cat. # 9271), anti-AKT (Cat. # 4691), anti-phospho-ERK (Cat. # 9101), and anti-RhoGDI1 (Cat # 2564) from Cell Signaling Technologies (Danvers, MA, USA); anti-Tiam1 (Cat. # AF5038) from R&D Systems (Minneapolis, MN, USA); anti-β2-chimaerin (Cat. # SAB4200059) from Sigma-Aldrich (St. Louis, MO, USA); anti Ly-GDI (D7) (sc-271108) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following antibodies, obtained from Sigma-Aldrich, were used at 1:5000 dilution: anti-actin (Cat. # A544) and anti-vinculin (Cat. # V9131). The following secondary antibodies were used at 1:3000 dilution: goat anti-mouse (Cat. # 172-1011), goat anti-rabbit (Cat. # 170-6515), and goat anti-sheep (Cat. # 172-1017) from BioRad (Hercules, CA, USA); and goat anti-rat (Cat. # 112-035-062) from Jackson ImmunoResearch (West Grove, PA, USA). Bands were visualized using enhanced chemiluminescence (ECL) with the LI-COR Odyssey Fc dual mode imaging system. 4.8. Analysis of mRNA Expression by qPCR mRNA expression levels were analyzed using reverse transcription qPCR. Briefly, total RNA was extracted using the RNeasy kit (QIAGEN, Valencia, CA, USA) and cDNA produced using the Taqman reverse transcription reagent (N8080234, Applied Biosystems, Waltham, MA, USA). qPCR amplifications were performed using a QuantStudio 3 detection system using carboxyfluorescein (FAM) tagged probes (ThermoFisher, Waltham, MA, USA). The FAM signal was normalized to endogenous β2-microglobulin housekeeping gene and fold-change calculated from comparative Ct analysis. 4.9. Migration and Invasion Assays Migration assays were performed with a Boyden chamber, using membranes with 12 μm pores and 5% FBS in the lower chamber as a chemoattractant. Invasion assays were performed in the same way but after coating of the upper side of the membrane with 0.5 mg/mL Matrigel. Experiments were carried out 48 h after transfection of RNAi duplexes, including a 24 h serum starvation prior to the assay, which was performed as previously described [ 53 ]. 4.10. In silico Analysis of P-REX1 mRNA Expression among Normal and Prostate Cancer Tissues Processed P-REX1 mRNA expression levels (Log2 transformed) and clinicopathological data from five independent prostate cancer studies were obtained from the CancerTools resource ( http://genomics.cicbiogune.es/CANCERTOOL/ ) [ 56 ]. Univariate and survival analysis were performed with R software. 4.11. Statistical Analysis Statistical analysis was done with student’s t -test or ANOVA using GraphPad Prism 3.0. A p -value <0.05 was considered statistically significant. Statistical significance of Kaplan–Maier curves was tested with the Mantel–Cox test. 5. Conclusions The present study demonstrates that Rac1 hyperactivation occurs in aggressive androgen-independent prostate cancer, and that this is independent of P-Rex1 Rac-GEF activity. Furthermore, this Rac1 hyperactivation was found to be insensitive to overexpression of the Rac-GAP β2-chimaerin. Our study also determined that increases in intracellular calcium lead to a rapid decrease in both Rac1-GTP and Cdc42-GTP levels. Supplementary Materials The following are available online at https://www.mdpi.com/2072-6694/12/2/480/s1 , Figure S1: Rac1-GTP and Cdc42-GTP levels in the androgen dependent VCaP cells and the effect of PI3K and Gβγ inhibition and Vav2/3 silencing in DU145 and PC3 cells, Figure S2: Association of β2-chimaerin expressed in PC3 and MCF-7 cells with a constitutively active Rac1 Q61L mutant and insensitivity of Rac1-GTP in PC3 cells to Rho activation, Figure S3: Expression of Rac-GEFs in PC3 cells, Figure S4: Silencing ALS2, TIAM2 or DOCK4 does not affect Cdc42-GTP levels, Figure S5: SmgGDS, Grb2 and IQGAP1 silencing have no effect on Rac1-GTP levels, Table S1: Proteomics of Rac1G15A binding proteins. Author Contributions Conceptualization, M.J.B., M.C.A., R.G.-M. and M.G.K.; Formal Analysis, M.J.B. and M.C.A.; Investigation, M.J.B.; Resources, R.G.-M. and M.G.K.; Writing—Original Draft Preparation, M.J.B. and M.G.K.; Writing—Review and Editing, M.J.B. and M.G.K.; Supervision, M.G.K.; Project Administration, M.G.K.; Funding Acquisition, M.J.B., R.G.-M. and M.G.K. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by National Institutes of Health, grant number R01-CA189765; National Institutes of Health, grant number R01-CA196232; National Institutes of Health, grant number R01-ES026023; National Institutes of Health, grant number 1R03CA234693-01; U.S. Department of Defense, grant number W81XWH1810274; Argentine National Agency of Scientific and Technological Promotion, grant number PICT-2015-0149. Conflicts of Interest The authors declare no conflict of interest. References Siegel, R.L.; Miller, K.D.; Jemal, A. 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Cancertool: A visualization and representation interface to exploit cancer datasets. Cancer Res. 2018 , 78 , 6320–6328. [ Google Scholar ] [ CrossRef ][ Green Version ] Figure 1. Rac1 and Cdc42 are hyperactivated in aggressive androgen-independent prostate cancer cells. ( A ) Representative Western blots of PAK-binding domain (PBD) pull-down assays showing Rac1-GTP and Cdc42-GTP in cells grown in the presence of 10% FBS. ( B ) Similar experiments were carried out after 24 h serum starvation. ( C , D ) Densitometric analysis of Rac1-GTP/total Rac1 and Cdc42-GTP/total Cdc42. Results normalized to LNCaP cells are expressed as the mean ± S.E. ( n = 4–5). ( E ) Active Rac1 and Cdc42 fractions in PC3 cells growing in the presence or absence of 10% FBS. Results are expressed as the mean ± S.E. ( n = 7) of the percentage of total Rac1 or Cdc42. * p < 0.05. ( F ) Representative Western blots of RhoGDI1 and RhoGDI2 expression in prostate cell lines. ( G ) Representative Western blot of Rac1b expression in prostate cancer cell lines. Figure 2. Rac1 hyperactivation in prostate cancer cells is independent of P-Rex1. ( A ) PC3 cells were transfected with P-Rex1, Tiam1 or non-target control ( NTC ) RNAi duplexes for 48 h, including a 24 h serum starvation prior to Rac1-GTP levels being determined using a PBD pull-down assay. Left panel , representative experiment. Right panel , densitometric analysis of Rac1-GTP levels normalized to total Rac1. Results, relative to NTC, are expressed as the mean ± S.E. ( n = 6). ( B ) Effect of the PI3K inhibitor BKM120 (2 μM, 60 min) on basal Rac1-GTP levels in DU145 and PC3 prostate cells, and on HRG-stimulated (20 ng/mL, 5 min) MCF-7 breast cancer cells. ( C ) Effect of the Gβγ inhibitor gallein (10 μM, 30 min) on basal Rac1-GTP levels in DU145 and PC3 prostate cells, and on HRG-stimulated (20 ng/mL, 5 min) MCF-7 breast cancer cells. For B and C, lower panels are representative experiments. Densitometric analyses of Rac1-GTP levels normalized to total Rac1 are expressed as the mean ± S.E. ( n = 3–4) relative to non-treated cells (PC3 and DU145) or HRG-stimulated cells (MCF-7). ( D ) Representative Western blot of P-Rex1 expression in PC3 cells subjected to P-Rex1 silencing for 48 h using two different RNAi duplexes (#1 and #2). ( E ) PC3 migration assay using a Boyden chamber without Matrigel. ( F ) PC3 invasion assay using a Boyden chamber with Matrigel. For ( E , F ), representative experiments are shown ( right panels ). A quantitative analysis of migration and invasion is shown in the left panels . Results normalized to parental cells (P) are expressed as the mean ± S.E. ( n = 3). * p < 0.05; *** p < 0.001. Figure 2. Rac1 hyperactivation in prostate cancer cells is independent of P-Rex1. ( A ) PC3 cells were transfected with P-Rex1, Tiam1 or non-target control ( NTC ) RNAi duplexes for 48 h, including a 24 h serum starvation prior to Rac1-GTP levels being determined using a PBD pull-down assay. Left panel , representative experiment. Right panel , densitometric analysis of Rac1-GTP levels normalized to total Rac1. Results, relative to NTC, are expressed as the mean ± S.E. ( n = 6). ( B ) Effect of the PI3K inhibitor BKM120 (2 μM, 60 min) on basal Rac1-GTP levels in DU145 and PC3 prostate cells, and on HRG-stimulated (20 ng/mL, 5 min) MCF-7 breast cancer cells. ( C ) Effect of the Gβγ inhibitor gallein (10 μM, 30 min) on basal Rac1-GTP levels in DU145 and PC3 prostate cells, and on HRG-stimulated (20 ng/mL, 5 min) MCF-7 breast cancer cells. For B and C, lower panels are representative experiments. Densitometric analyses of Rac1-GTP levels normalized to total Rac1 are expressed as the mean ± S.E. ( n = 3–4) relative to non-treated cells (PC3 and DU145) or HRG-stimulated cells (MCF-7). ( D ) Representative Western blot of P-Rex1 expression in PC3 cells subjected to P-Rex1 silencing for 48 h using two different RNAi duplexes (#1 and #2). ( E ) PC3 migration assay using a Boyden chamber without Matrigel. ( F ) PC3 invasion assay using a Boyden chamber with Matrigel. For ( E , F ), representative experiments are shown ( right panels ). A quantitative analysis of migration and invasion is shown in the left panels . Results normalized to parental cells (P) are expressed as the mean ± S.E. ( n = 3). * p < 0.05; *** p < 0.001. Figure 3. Expression of P-Rex1 in prostate cancer. ( A ) mRNA expression level of P-Rex1 in the indicated prostate cancer cell lines was assessed by RT-qPCR. ( B ) Violin plots showing the status of P-Rex1 mRNA expression in prostate cancer tumor and normal tissues. ( C ) Violin plots showing P-Rex1 mRNA expression relative to Gleason score. ( D ) Kaplan–Maier curves showing the disease-free survival of patient groups selected according to the PREX1 quartile expression. Analysis was performed using publicly available datasets from ( B ) Grasso et al. [ 31 ], Lapointe et al. [ 32 ], and Varambaly et al. [ 33 ]. ( B , D ) Taylor et al. [ 34 ] and ( D ) TCGA. Figure 4. Elevated Rac1 activation in androgen-independent prostate cancer cells is insensitive to the Rac-GAP β2-chimaerin. ( A ) DU145 and PC3 cells were infected with increasing MOIs (0–100 pfu/cell) of a β2-chimaerin AdV. After 16 h, Rac1-GTP levels were determined using a PBD pull-down assay. ( B ) Similar experiments were carried out in MCF-7 cells stimulated with HRG (20 ng/mL, 5 min). Left panels , representative experiments. Right panels , densitometric analyses of Rac1-GTP/total Rac1. Results (normalized to MOI = 0 pfu/cell) are expressed as the mean ± S.E. ( n = 3–4). , p < 0.05; n.s., not significant. Figure 4. Elevated Rac1 activation in androgen-independent prostate cancer cells is insensitive to the Rac-GAP β2-chimaerin. ( A ) DU145 and PC3 cells were infected with increasing MOIs (0–100 pfu/cell) of a β2-chimaerin AdV. After 16 h, Rac1-GTP levels were determined using a PBD pull-down assay. ( B ) Similar experiments were carried out in MCF-7 cells stimulated with HRG (20 ng/mL, 5 min). Left panels , representative experiments. Right panels , densitometric analyses of Rac1-GTP/total Rac1. Results (normalized to MOI = 0 pfu/cell) are expressed as the mean ± S.E. ( n = 3–4). , p < 0.05; n.s., not significant. Figure 5. G-LISA screen of Rac-GEFs. ( A ) PC3 cells with stable depletion of indicated Rac-GEFs were serum starved and Rac1-GTP levels analyzed using a G-LISA assay screen. Rac1-GTP levels, normalized to total Rac1 assessed by Western blot and expressed relative to NTC1, are expressed as the mean of three replicates ± S.D. ( B ) PC3 cells were transfected with siRNA duplexes targeting ALS2, TIAM2, DOCK4 or a non-target control ( NTC ), and Rac1-GTP levels measured using a PBD assay after 48 h. Left panel , representative experiment. Right panel , densitometric analysis of Rac1-GTP/total Rac1. Results, relative to parental cells, are expressed as the mean ± S.E. ( n = 3). Figure 5. G-LISA screen of Rac-GEFs. ( A ) PC3 cells with stable depletion of indicated Rac-GEFs were serum starved and Rac1-GTP levels analyzed using a G-LISA assay screen. Rac1-GTP levels, normalized to total Rac1 assessed by Western blot and expressed relative to NTC1, are expressed as the mean of three replicates ± S.D. ( B ) PC3 cells were transfected with siRNA duplexes targeting ALS2, TIAM2, DOCK4 or a non-target control ( NTC ), and Rac1-GTP levels measured using a PBD assay after 48 h. Left panel , representative experiment. Right panel , densitometric analysis of Rac1-GTP/total Rac1. Results, relative to parental cells, are expressed as the mean ± S.E. ( n = 3). Figure 6. Increased Rac1-GTP levels in PC3 cells are sensitive to elevations in intracellular calcium. ( A ) Serum starved PC3 cells were stimulated with FBS (10%) for the indicated time and a PBD pull-down assay was performed to determine Rac1-GTP and Cdc42-GTP levels. ( B ) Similar experiments were carried out in LNCaP cells. For ( A , B ) Left panels , representative experiments. Right panels , densitometric analysis of Rac1-GTP levels normalized to total Rac1, and Cdc42-GTP levels normalized to total Cdc42. ( C – F ) Serum starved PC3 cells were treated with DMSO (0.1%), A23187 (2 μM), thapsigargin (1 μM) or forskolin (1 μM) for the indicated times, and PBD pull assays were performed to determine Rac1-GTP and Cdc42-GTP levels. ( C – F ) Representative experiments are shown for ( C ) DMSO, ( D ) A23187, ( E ) thapsigargin and ( F ) forskolin treatment. ( G ) Densitometric analysis of Rac1-GTP levels normalized to total Rac1. Results, relative to the unstimulated time point, are expressed as the mean ± S.E. ( n = 3–7). * p < 0.01; * p < 0.001; ** p < 0.0001. Figure 6. Increased Rac1-GTP levels in PC3 cells are sensitive to elevations in intracellular calcium. ( A ) Serum starved PC3 cells were stimulated with FBS (10%) for the indicated time and a PBD pull-down assay was performed to determine Rac1-GTP and Cdc42-GTP levels. ( B ) Similar experiments were carried out in LNCaP cells. For ( A , B ) Left panels , representative experiments. Right panels , densitometric analysis of Rac1-GTP levels normalized to total Rac1, and Cdc42-GTP levels normalized to total Cdc42. ( C – F ) Serum starved PC3 cells were treated with DMSO (0.1%), A23187 (2 μM), thapsigargin (1 μM) or forskolin (1 μM) for the indicated times, and PBD pull assays were performed to determine Rac1-GTP and Cdc42-GTP levels. ( C – F ) Representative experiments are shown for ( C ) DMSO, ( D ) A23187, ( E ) thapsigargin and ( F ) forskolin treatment. ( G ) Densitometric analysis of Rac1-GTP levels normalized to total Rac1. Results, relative to the unstimulated time point, are expressed as the mean ± S.E. ( n = 3–7). * p < 0.05; p < 0.01; * p < 0.001; **** p < 0.0001.	https://www.mdpi.com/2072-6694/12/2/480/html
Biomineralization of Uniform Gallium Oxide Rods with Cellular Compatibility \| Request PDF Request PDF \| Biomineralization of Uniform Gallium Oxide Rods with Cellular Compatibility \| Monodispersed single crystalline alpha-GaOOH rods coated by silk fibroin (SF) have been prepared via a facile biomineralization process in the... \| Find, read and cite all the research you need on ResearchGate Biomineralization of Uniform Gallium Oxide Rods with Cellular Compatibility July 2009 Inorganic Chemistry 48(14):6471-9 DOI: 10.1021/ic900420m Authors: Guangfu Yin Sichuan University Xiaoming Liao Abstract Monodispersed single crystalline alpha-GaOOH rods coated by silk fibroin (SF) have been prepared via a facile biomineralization process in the template of SF peptide. The carbon-coated alpha-Ga(2)O(3) and beta-Ga(2)O(3) rods are obtained by thermal treatment of the alpha-GaOOH rods at 600 and 800 degrees C, respectively. In vitro cytotoxicity studies of these gallium oxide rods showed no significant effect leading to restraint of cell proliferation of L929, Hela, and HaCat cells in less than 0.1 mg/mL prepared rods. On the basis of their excellent luminescence emission properties and cellular compatibilities, possible applications for bio-optoelectronic devices can be envisioned. ... It has attracted attention for its capability for high current, high speed switching with low loss, which is a prerequisite in ultraviolet (UV) transparent electrodes and solar-blind ultraviolet photodetectors. [1][2][3] [4] [5] Gallium oxide is known to exhibit six crystal structures, including a monoclinic (β), a corundum (α), a spinel (γ), two orthorhombic (ε and δ), and a transient polymorph κ phase, which is an analog of orthorhombic-Al 2 O 3 . 6,7 β-Ga 2 O 3 was the dominant focus of early research because it was possible to grow high crystalline thin films in thermodynamic stability, but the substrate required expensive processes and it was challenging to use large-area fabrication. ... ... Except for the 350 nm peak, all other peaks are similar to those previously reported. 4, [32][33][34][35] The state transition from these six commonly observed peaks may come from native point defects such as oxygen and gallium vacancy. 4,[32][33][34][35] The origin of the 350 nm peak from CL data was previously reported on hydride vapor phase epitaxy (HVPE) grown Sn-doped α-Ga 2 O 3 . ... ... 4,[32][33][34][35] The state transition from these six commonly observed peaks may come from native point defects such as oxygen and gallium vacancy. 4, [32][33][34][35] The origin of the 350 nm peak from CL data was previously reported on hydride vapor phase epitaxy (HVPE) grown Sn-doped α-Ga 2 O 3 . 33 was implemented, and the 350 nm peak in this study originates from a different defect state. ... Luminescence properties related anti-phase domain of alpha-Ga2O3 Duyoung Yang Young-Woon Kim This work investigates the relationship between atomic arrangement and luminescence properties in a high-quality alpha-Ga2O3 thin film grown on an Al2O3 single-crystal membrane. The strain induced by merging domain boundaries shows more significant variability in annular darkfield images even though there is no additional gallium concentration confirmed. The bandgap energy of alpha-Ga2O3 is estimated to be 5.56 eV from the CL measurement in a transmission electron microscope. A peak at 320 nm was observed within the domain, while the domain boundary showed spectrum peaks with 380–480 nm. The anti-phase domain (APD) is formed by the instabilities of Al–O bonding templates provided by the Al2O3 substrate. The APD boundary gives a characteristic wavelength of 350 nm, which is the result of the merging boundary of in-phase and anti-phase domains. ... The photoluminescence (PL) properties of as-prepared ZnO were investigated. Based on its unique luminescence properties, it is of great significance to explore the biological performance of nanoscaled ZnO for possible applications in the biomedicine field [32, 33]. It has not been revealed that nanoscaled ZnO could show both good cytocompatibility and good antimicrobial activity. ... Nanoscaled Zinc Oxide Prepared by Mono-amino Acid Templated Assembly and Their Superior Biological Properties Article Full-text available Mar 2022 J CLUST SCI Chengfang Tan Zhiying Sun Ying Ruan Hui Liu Nanoscaled zinc oxide (ZnO) was synthesized via a benign mineralization reaction using mono-amino acid as templates. Characterized by Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy, it was found that the microstructure of as-prepared ZnO can be fine-tuned into olive-like and flake-like structures with leucine and histidine respectively. Otherwise, the photoluminescence (PL) results indicate that biomineralized ZnO nanoparticles show a potential application in the fields of optical devices. To further explore the biological performance of as-prepared ZnO nanoparticles, antibacterial activity tests against Staphylococcus aureus and Escherichia coli, and cytocompatibility on mouse MC3T3 cells were also investigated. The superior biological properties of these biomineralized ZnO together with their intrinsic photoluminescent properties open perspectives for the development of biomedical applications. ... According to the literature, the bandgap energy of gallium oxyhydroxide, determined by UV-vis spectroscopy, is between 4.75 and 5.06 eV; whereas for gallium oxide spans from 4.8 to 4.9 eV. [29][30] [31] [32][33] In this work, the bandgap energy value obtained for GaOOH and α-Ga 2 O 3 was the same for both compounds (5 eV) and is consistent with the literature (Figure 6). A gallium oxide sample taken after the degradation of malachite green was analyzed, and a slight reduction of the bandgap energy to 4.5 eV was measured (Figure 6c). ... α-Ga 2 O 3 as a Photocatalyst in the Degradation of Malachite Green Article Full-text available Claudia Isabel Macias Rodríguez Miguel Ángel López Álvarez α-Ga2O3 is a wide-bandgap semiconductor material which was prepared by a novel synthesis method from metallic gallium. It was characterized by X-ray diffraction, infrared spectroscopy, ultraviolet spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. This oxide was also evaluated as a photocatalyst toward the decomposition of malachite green (MG). X-ray photoelectron spectroscopy was used with the purpose of analyzing the changes on the surface of the material before and after the photocatalytic reaction. The results found by X-ray diffraction shows that alfa phase did not transform to another crystalline phase during the reaction. However, a slight change on the relative intensities of the planes (104) and (110), may explain the variation of the morphology of the oxide, associated to a preferential particle erosion. High resolution XPS analyses revealed a shift toward lower binding energies of the O1s level after the photocatalytic reaction, suggesting the presence of oxygen bound to carbonyls or alcohols. Organic nitrogenous residues associated to MG were also detected by the presence of the N1s band observed at 396.7 eV. © The Author(s) 2019. Published by ECS. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/2.0351907jss] ... Among the different materials susceptible to be used as DBR-based microcavities, monoclinic β-Ga 2 O 3 is of high interest. It is a transparent conductive oxide (TCO) that has attracted strong interest due to its high chemical and thermal stability, wide transparency range (E g ≈ 4.9 eV) [17], controllable conductivity [18], excellent properties for high-power electronics due to its very high breakdown field [19,20] as well as its biocompatibility [21, 22]. ... Modal Analysis of β − Ga 2 O 3 : Cr Widely Tunable Luminescent Optical Microcavities Jose San Juan Bianchi Mendez Optical microcavities are key elements in many photonic devices, and those based on distributed Bragg reflectors (DBRs) enhance dramatically the end reflectivity, allowing for higher quality factors and finesse values. Besides, they allow for wide wavelength tunability, needed for nano- and microscale light sources to be used as photonic building blocks in the micro- and nanoscale. Understanding the complete behavior of light within the cavity is essential to obtaining an optimized design of properties and optical tunability. In this work, focused ion-beam fabrication of high refractive-index contrast DBR-based optical cavities within Ga2O3:Cr microwires grown and doped by the vapor-solid mechanism is reported. Room-temperature microphotoluminescence spectra show strong modulations from about 650 nm up to beyond 800 nm due to the microcavity resonance modes. Selectivity of the peak wavelength is achieved for two different cavities, demonstrating the tunability of this kind of optical system. Analysis of the confined modes is carried out by an analytical approximation and by finite-difference–time-domain simulations. A good agreement is obtained between the reflectivity values of the DBRs calculated from the experimental resonance spectra, and those obtained by finite-difference–time-domain simulations. Experimental reflectivities up to 70% are observed in the studied wavelength range and cavities, and simulations demonstrate that reflectivities up to about 90% could be reached. Therefore, Ga2O3:Cr high-reflectivity optical microcavities are shown as good candidates for single-material-based, widely tunable light emitters for micro- and nanodevices. ... Ga 2 O 3 , especially its stable monoclinic form is an important metal-oxide semiconductor having a wide band gap (E g ) of 4.9 eV exhibiting versatile range of applications. Novel structures of b-Ga 2 O 3 possess peculiar physical and chemical properties that are useful in optoelectronic devices, gas sensors, heterogeneous catalysis, solar cells, blue light emitter, luminescent phosphors [3][4][5], etc. Due to the versatile properties and wide applications, Ga 2 O 3 nanorods, nanowires have been synthesized by a variety of fabrication techniques, which includes hydrothermal method, sol-gel technique, forced hydrolysis, sonochemical process, microwave-assisted heating, biomineralization process, precipitation technique and so forth [6][7][8] [9] . Nanorods provide fundamental scientific opportunity for investigating the influence of size and dimensionality with respect to other novel properties and applications resulting from the orientation and arrangement of nanoparticles. Aligned nanorods of b-Ga 2 O 3 have not been explored until now, which has hindered experimental investigation of the morphology dependent properties. ... Morphology controllable synthesis of parallely arranged single-crystalline β-Ga2O3 nanorods for photocatalytic and antimicrobial activities CHEM ENG J K. Girija S. Thirumalairajan Devanesan Mangalaraj ... The biomineralization, a facile room-temperature aqueous mineralization process, has been developed. During the process, the natural biomineralization conditions could be mimicked, and biotemplates (proteins, peptides, or DNA) are used as the structure-directing agent to control the nucleation and yield a unique crystal structure [12] [13] . It is known that the progression of tumor cells requires complex interactions with the host tissues. ... Inhibiting the motility and invasion of cancer cells by biomineralization Guangfu Yin Chuying Ma Yadong Yao ... Obviously, it is urgent to provide some simple and mild methods to prepare the NPs coated with peptides. In recent years, a facile room-temperature aqueous mineralization process, biomineralization, has been developed [49, 50]. During the process, the natural biomineralization process conditions could be mimicked, and biotemplates (proteins, peptides or DNA) are used as the structure-directing agent to control the nucleation and yield a unique crystal structure. ... Synthesis and cellular compatibility of biomineralized Fe3O4 nanoparticles in tumor cells targeting peptides Guangfu Yin Chuying Ma Hao Yin Origin of extra diffraction spots for high crystalline alpha-Ga 2 O 3 Feb 2023 Yong-Hee Lee Duyoung Yang Byeongjun Gil Young-Woon Kim This work has investigated the microstructure characteristics of high-quality alpha-Ga 2 O 3 thin film grown on the Al 2 O 3 single crystal substrate membrane. Hetero-epitaxial alpha Ga 2 O 3 crystals reveal the formation of a three-fold symmetry at the initial stage of the growth by the oxygen template provided by the Al 2 O 3 . Inversion domains are found, and they have a 180° inverted configuration from the surroundings. These IDs lead to extra diffraction spots when observed along [110] and [010]. Polymorphism and Faceting in Ga 2 O 3 Layers Grown by HVPE at Various Gallium‐to‐Oxygen Ratios Nov 2021 Sevastian Shapenkov Oleg Vyvenko Vladimir Nikolaev Georgiy Varygin Herein, the influence of the oxygen flow on the formation of metastable polymorphs of gallium oxide (Ga2O3) grown by halide vapor phase epitaxy (HVPE) on c-plane patterned sapphire substrates (PSS), on gallium nitride (GaN) templates, and on m-plane smooth sapphire substrates is reported. X-ray diffraction, scanning electron microscopy, and cathodoluminescence are used to identify different polymorphic phases. The samples deposited on bare PSS exhibit faceted growth of the α-Ga2O3 on the cones of the sapphire substrate and the formation of the κ-Ga2O3 between the cones. In contrast, growth on GaN templates results in hexagonal columns of κ-Ga2O3 which produce a continuous smooth layer upon coalescence. The growth of Ga2O3 on m-plane sapphire substrates results in overgrown pyramids of the α-phase. For all types of substrates, the variation of the oxygen flow affects only the thickness and coalescence of Ga2O3 layers. Thus, the growth of Ga2O3 metastable polymorphs is mainly influenced by the form, orientation, and symmetry of the substrate and shows a weak dependence on stoichiometry. View HVPE α‐ and ε‐Ga 2 O 3 Epitaxial Films Grown on Patterned Sapphire Substrates Sevastian Shapenkov Oleg Vyvenko Evgeny Ubyivovk Vladimir Nikolaev We report on the growth of Ga2O3 films by halide vapor phase epitaxy on plain and cone‐shape‐patterned sapphire substrates (PSS). The obtained specimens were characterized by x‐ray diffraction, transmission electron microscopy, cathodoluminescence, optical transmission spectroscopy, and current‐voltage measurements. Both types of Ga2O3 layers were of a reasonably high crystal quality; their physical properties, however, were very different. Under the same conditions, the growth on plain substrates resulted in a continuous α‐Ga2O3 layer, while the growth on PSS produced a regular array of α‐Ga2O3 columns on top of sapphire cones with the space between them filled with ε‐Ga2O3. Ga2O3 films grown on plain sapphire were insulating; by contrast, Ga2O3 films grown on PSS were conducting. It was found that the conductivity of Ga2O3 on PSS follows the Arrhenius law with the activation energy of 0.33 eV. New luminescent bands for α‐ and ε‐phases have been found. Spectral components of the defect‐related luminescence for α‐ and ε‐ phases have been identified. This article is protected by copyright. All rights reserved. Morphology controllable synthesis and characterization of gallium compound hierarchical structures via forced-hydrolysis method J ALLOY COMPD Bongkyun Kang Guh-Hwan Lim Byungkwon Lim Dae Ho Yoon To our best knowledge, β-Ga2O3 via hierarchical gallium precursors from nanoparticles to microrods was successfully synthesized for first time using the forced-hydrolysis method and the calcination process. We report the growth mechanism and formation of various morphological gallium precursors such as Ga4(OH)10SO4 nanospheres and ɑ-GaOOH microrods. The influence of the growth time on the crystal structure and its morphology was studied. The results indicate that the growth time played an important role in the transformation from amorphous to single-crystal structures. After calcination at 1000 °C for 1 hour, well-crystallized and porous β-Ga2O3 microrods are formed. The enhanced broad blue emission of porous β-Ga2O3 microrods were related to defect band emission, such as oxygen vacancies and gallium-oxygen vacancy pairs. We propose that this oriented growth and transformation model may also be applicable to crystal growth and phase transformations and support the fabrication of other three-dimensional hierarchical materials, which is potentially important for a broad range of optoelectronic applications. Interfaces with Tunable Mechanical and Radiosensitising Properties Article Feb 2016 ACS APPL MATER INTER Nora G. Berg Brady L. Pearce Patrick Snyder Albena Ivanisevic We report the fabrication of a composite containing nanostructured GaOOH and MatrigelTM with tunable radiosensitising and stiffness properties. Composite characterization was done with microscopy and rheology. The utility of the interface was tested in vitro using fibroblasts. Cell viability and reactive oxygen species assays quantified the effects of radiation dosages and GaOOH concentrations. Fibroblasts' viability decreased with increasing concentration of GaOOH and composite stiffness. During ionizing radiation experiments the presence of the scintillating GaOOH triggered a different cellular response. Reactive oxygen species data demonstrated that one can reduce the amount of radiation needed to modulate the behavior of cells on interfaces with different stiffness containing a radiosensitising material. Synthesis and optical properties of GaOOH and Ga2O3 Article Full-text available May 2013 Jianjie Liang X.-B. Wang Yalian Zhang B.-S. Xu GaOOH precursors with different morphologies and sizes were synthesized by hydrothermal method. The products were characterized by XRD, SEM and the probable growth mechanism of GaOOH was analyzed. The pH values played a dominated role in adjusting GaOOH morphologies and had an effect on their crystallizations. The GaOOH precursors were calcined in air and transformed into α-Ga2O3 and β-Ga2O3 under 600°C and 900°C, respectively. The samples after calcinations showed coincident morphology hereditability. Photoluminescence measurements(λex=250 nm) were conducted for the GaOOH and Ga2O3 samples and the emission peaks located at 415 nm and 465 nm when pH=5, however, at 370 nm and 465 nm for pH=3 and 8. Fabrication of Gd/Eu-codoped SmPO4 Nanorods for Dual-modal Magnetic Resonance and Bio-optical Imaging Article Oct 2015 J COLLOID INTERF SCI Zhi Wu Zhongbing Huang Guangfu Yin Fabao Gao Ln-based complexes can be used as T1-enhanced contrast agents of magnetic resonance (MR) imaging in clinical field. Herein, we present a facile and feasible biomineralization process to fabricate Gd/Eu-codoped SmPO4 nanorods (NRs) with silk fibroin (SF) peptides (codoped SF-NRs) as T1-enhanced contrast agents, which possess paramagnetic property, photoluminescence (PL), better cyto-/tissue-compatibility and longer half-life in blood due to SF coating on their surface. Their bio-distributions in TB-N mice via tail-vein injection indicated that, although SF-NRs could be safely cleared away through renal and fecal excretion, SF-NRs easily permeated and aggregated in tumors. The results of in vitro MR imaging demonstrate that the longitudinal relaxivity r1 value of codoped SF-NRs (0.31Sm-GdmM(-1)s(-1)) is not only significantly higher than those of Gd-doped and Eu-doped SmPO4 SF-NRs, but also higher than those of codoped pure NRs. The tests of in vivo T1 weighted MR imaging via intro-tumor injection and tail-vein injection confirm that, compared to the pure NRs, the codoped SF-NRs exhibited higher positive signal-enhancement ability. Furthermore, the better luminescence imaging of living cells under the fluorescence microscope (94% stronger than that of the NRs without SF). A formation mechanism of codoped SF-NRs is proposed, to explain the synergistic effect of Gd/Eu codoping and SF coating on their enhanced bio-compatibility, half-life in blood, T1-weighted MR imaging and PL imaging. Water-Soluble Thin Film Transistors and Circuits Based on Amorphous Indium–Gallium–Zinc Oxide Article Mar 2015 ACS APPL MATER INTER Sung Hun Jin Seung-Kyun Kang In-Tak Cho John A. Rogers This paper presents device designs, circuit demonstrations, and dissolution kinetics for amorphous indium-gallium-zinc-oxide (a-IGZO) thin film transistors (TFTs) comprised completely of water-soluble materials, including SiNx, SiOx, molybdenum and polyvinyl alcohol (PVA). Collections of these types of physically transient a-IGZO TFTs and 5-stage ring oscillators (ROs) constructed with them, show field effect mobilities (~10 cm2/Vsec), on/off ratios (~2x10^6), sub-threshold slopes (~220 mV/dec), Ohmic contact properties and oscillation frequency of 5.67 kHz at supply voltages of 19 V, all comparable to otherwise similar devices constructed in conventional ways with standard, non-transient materials. Studies of dissolution kinetics for a-IGZO films in deionized. water, bovine serum, and phosphate buffer saline solution provide data of relevance for the potential use of these materials and this technology in temporary biomedical implants. Facile preparation and bifunction imaging of Eu-doped GdPO4 nanorods with MRI and cellular luminescence Article Jan 2015 DALTON T Qijun Du Zhongbing Huang Zhi Wu Lei Wang The biocompatibility of nanomaterials with multi-functions is very important for their clinical applications. Herein, the hexagonal crystal Eu−doped GdPO4 nanorods (NRs) in the template of silk fibroin (SF) peptides are successfully synthesized via a mineralization process. The sizes of the Eu-doped GdPO4 NRs with SF peptides (SF-NRs) are the length of ~150 nm and the diameter of ~10 nm. The Eu-doped SF-NRs have strong pink luminescence and the mass magnetic susceptibility value (1.27 emu/g in 20000 G of magnetic field) due to Eu ions doping. The cell test indicates that the Eu-doped SF-NRs obviously promote the viability of cells at NRs concentration of 25-200 g/mL. A growth mechanism of Eu-doped GdPO4 SF-NRs is proposed, to explain their strong cellular luminescence, magnetic resonance (MR) imaging and good cyto-compatibility. Compared to NRs without SF, the Eu-doped SF-NRs not only exhibit a higher effective positive signal-enhancement ability (the longitudinal relaxivity r1 value is 1.38 (Gd mM∙s)-1) and in vivo T1 weighted MR imaging enhancement under a 7.0 T MRI system, but also show the better luminescence imaging of living cells under the fluorescence microscope, indicating that the Eu-doped SF-NRs have potential as T1 MRI contrast agents and optical imaging probe. Structure, Property, and Function of Gallium/Urea and Gallium/Polyethylene Glycol Composites and Their Sintering Products: β- and γ-Gallium Oxide Nanocrystals Article Oct 2012 Le Xin Song Yue Teng Jie Chen The first part of the study is devoted to the comparison between the doping effect of urea (a small molecule) and polyethylene glycol (PEG, a long-chain polymer) on the physical property of metallic gallium (Ga). The physical properties of the Ga composited in the two materials, Ga/urea and Ga/PEG, were investigated by scanning electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, differential scanning calorimetry, superconducting quantum interference device, and surface-enhanced Raman scattering spectra and compared with our previous results for the effect of macrocyclic hosts (e.g., cyclodextrins, calixarenes) on the physical modification of metallic Ga. Our data provide new direct evidence that the modification of physical properties of Ga is highly dependent on the nature of dopants used. For example, the addition of a small amount of urea causes a fundamental change in the crystallization behavior of Ga, and the presence of PEG results in the occurrence of a weak paramagnetism of Ga at high fields, both of which are completely different from the effect of other dopants. The other part of the study is devoted to demonstrating whether there is a significant difference in the oxidation process of metallic Ga and its composites. Our result gives a strong positive answer to the question. β- and γ-gallium oxide nanocrystals were obtained by sintering the Ga/urea composite at different temperatures and exhibited distinctive photoluminescence and photocatalysis properties. These results gave a strong impression that the introduction of different dopants leads metallic Ga to generate different features in microstructure, physical property, and especially chemical reactivity. We believe that the findings of this study have important implications for the development of inorganic materials. Fabrication and neuron cytocompatibility of iron oxide nanoparticles coated with silk-fibroin peptides Article Jan 2014 Min Deng Zhongbing Huang Yuanwen Zou Jianwen Gu Neuro-cytotoxicity of iron oxide nanoparticles (NPs) need to be considered. In this paper, magnetic Fe3O4 NPs are synthesized via a bio-mineralization process using silk fibroin (SF) as template. The prepared NPs with SF (SF-NPs) have more uniform grain size, better stability in weakly alkaline solution and higher saturation magnetization (about 82emu/g) than pure NPs. After exposure to different concentration of SF-NPs (6.25-100μg/mL), the reactive oxygen species generation in PC12 cells reduced compared with pure NPs. 1-5d treatment with SF-NPs did not destroy cell membrane and cyto-skeleton, and could improve the neurites extension in a dose-dependent manner at lower concentration (6.25-50μg/mL), because SF peptide coating could delay the release of iron ions and the increase of surface crystal defects of NPs. Intact mitochondria in a neurite indicate the extension activity of neurites of cells treated with SF-NPs. Their high magnetic property and good neural-cytocompatibility provide the possibility of SF-NPs to be used as the contrast agents in brain MRI or the carrier of neural therapeutic drugs. Preparation of LiNbO3 nanoparticles using poly(L-lysine) as a biomolecular additive Article Mar 2014 MATER CHEM PHYS Youngjoon Lim Sang-Yup Lee The effects of poly(l-lysine) as a biomolecular additive on the synthesis of LiNbO3 were investigated. PLL is a widely-studied biomolecule containing amino groups that can interact with solid inorganic clusters. The addition of PLL to a LiNbO3 precursor solution enhanced the aggregation of the produced LiNbO3 nanoparticles. This aggregation was induced by the electrical attraction of PLL with LiNbO3 nanoparticles, and was enhanced with increasing PLL molecular weight. Furthermore, the association of PLL with LiNbO3 nanoparticles was increased by the addition of methanol, which enhanced the miscibility of PLL with the precursor solution working as a co-solvent. The LiNbO3 nanoparticles generated with PLL exhibited piezoelectric properties without post-thermal treatment, suggesting that PLL contributes to the piezoelectricity. The results of this study are intriguing in terms of the potential for diverse engineering nanomaterials synthesis through a biomolecule that can also improve the physicochemical properties. Histidine-Assisted Synthesis and Cellular Compatibility of Magnetic Cobalt Oxide Nanoparticles at Room Temperature Article Mar 2011 Junjie Zhao Zhongbing Huang Jingwen Zeng Jianwen Gu Magnetic nanoparticles (NPs) of cobalt oxide (Co3O4) with the diameter of 20–40 nm have been prepared by a simple liquid deposition method in the Histidine (His) assistance at room temperature. Ethanol plays an important role in the preparation of cobalt oxide NPs with a polycrystalline structure. The growth mechanism for Co3O4 cube particles has been preliminarily explained. The hysteresis loop of NPs reveals their good magnetic property indicating that they can be used in hyperthermia, cell separation etc. These applications need the magnetic particles with cytocompatible properties. The analysis of IR spectrum, TG curve and HRTEM image indicated that cobalt oxide particles was conjugated with the His molecules. Escherichia coli (E. coli) and L929 cells tests suggest a good cellular compatibility at a concentration of less than 0.25 mg/mL, indicating that the prepared Co3O4 NPs have a potential for several biomedical applications. View Show abstract Preparation of ZnFe2O4 nanoparticles in the template of silk-fibroin peptide and their neuro-cytocompability in PC12 cells Article Feb 2013 Juan Liu Min Deng Zhongbing Huang Jianwen Gu There is an urgency to prepare non-neurotoxic nanoparticles (NPs) as MRI contrast agent. We prepared ZnFe2O4 NPs with monocrystal structure in the template of silk-fibroin (SF) peptide at room temperature. Analyses of their crystallite size and NPs diameter after the growth of 1d and 8d indicated that SF peptide could promote nucleation, growth and dispersion of NPs. Results of EDS, Raman and TG proved that there was 4wt% of the chemical composition of SF in as-prepared NPs. A possible growth mechanism of ZnFe2O4 NPs in the template of SF is proposed. The saturation magnetizations of NPs with SF (SF-NPs) were about 15 and 30emu/g for 1 and 8 d, respectively. SF-NPs obviously promoted the viability of PC12 cells at NPs concentration of 0.0625mg/mL in 2d and 5d of co-culture. Analysis of neurite length indicated no significant decreases of the experimental groups at different SF-NPs concentrations (from 0.0625 to 0.5mg/mL) after 5d cell co-culture, because SF peptide coating could slow the release of iron/zinc ions from NPs. Furthermore, SF-NPs did not destroy the organelles, karyotheca and the neurite using the observation in the cell ultrathin sections. Based on their good magnetic property and neuro-cytocompatibility, the potential application of ZnFe2O4 NPs with SF as MRI contrast agents would be envisioned. Dipeptide-assisted growth of uniform gallium oxohydroxide spindles Article Jul 2010 J CRYST GROWTH Inho Lee Jinyoung Kwak Seungjoo Haam Sang-Yup Lee The catalytic dipeptide His–Ser was used as an additive in mineralizing gallium ions to form GaOOH, a solid precursor of Ga2O3. This dipeptide was chosen to mimic the enzyme structure of silicatein, similar to the well-known catalytic triad of chymotrypsin. The dipeptide promoted formation of spindle-structured GaOOH under acidic conditions by behaving as a heterogeneous nucleation seed. In contrast, no well-defined, structured gallium species were produced in the absence of dipeptide. The catalytic function of the dipeptide was most pronounced at pH values in the range 3–5, which are lower than the pKa of imidazole in the His side chain. These results suggest that the catalytic role of dipeptide influences the gallium hydroxide conversion and growth. This study suggests that a designed peptide with active functionality can be further exploited to produce inorganic compounds with controlled nucleation and growth. Preparation and cell response of bio-mineralized Fe3O4 nanoparticles Article Jul 2011 J COLLOID INTERF SCI Guangfu Yin Zhongbing Huang Min Deng Jianwen Gu Silk fibroin (SF)-coated Fe(3)O(4) nanoparticles (NPs) with good superparamagnetism were successfully prepared via a bio-mineralization process at room temperature. Two cell tests revealed that mineralized SF-coated Fe(3)O(4) NPs presented good cytocompatibility for L929 and osteoblast cells and higher cell density after 5 d with high concentrations of SF-coated Fe(3)O(4) NPs (up to 0.5 mg/mL). These resulted from SF surface coating on NPs, nano-surface morphology and iron ion release of Fe(3)O(4) NPs. The mineralized SF-coated Fe(3)O(4) NPs could be envisioned for various bone orthopedic and therapeutic applications, in which SF-coated NPs location is controlled through an external magnetic field to promoted bone growth. Lanthanide-Ion-Assisted Structural Collapse of Layered GaOOH Lattice Article May 2011 INORG CHEM Sanyasi Naidu Boddu M. Pandey Jitendra Nuwad Chirakarumpil Gopalan Nair Sivan Pillai GaOOH nanorods were prepared by hydrolysis of Ga(NO(3))(3)·xH(2)O by urea at ~100 °C in the presence of different amounts of lanthanide ions like Eu(3+), Tb(3+), and Dy(3+). On the basis of X-ray diffraction and vibrational studies, it is confirmed that layered structure of GaOOH collapses even when very small amounts of lanthanide ions (1 atom % and more) are present in the reaction medium during the synthesis of GaOOH nanorods. The incorporation of lanthanide ions at the interlayer spacing of the GaOOH lattice, followed by its reaction with OH groups that connect the layers containing edge-shared GaO(6) in GaOOH, is the reason for the collapse of the layered structure and associated amorphization. This leads to the formation of finely mixed hydroxides of lanthanide and gallium ions. These results are further confirmed by steady-state luminescence and excited-state lifetime measurements carried out on the samples. The morphology of the nanorods is maintained upon heat treatment at high temperatures like 500 and 900 °C, and during this process, the finely mixed lanthanide and gallium hydroxides facilitate diffusion of lanthanide ions into the Ga(2)O(3) lattice, as revealed by the existence of strong energy transfer with an efficiency of more than 90% between the host and lanthanide ions. Synthesis of a mesoporous single crystal Ga2O3 nanoplate with improved photoluminescence and high sensitivity in detecting CO Article Sep 2010 CHEM COMMUN Shicheng‘ Yan Lijuan Wan Zhaosheng Li Zhigang Zou A new approach is proposed to synthesize a mesoporous single crystal Ga(2)O(3) nanoplate by heating a single crystal nanoplate of GaOOH, which involves an ion exchange between KGaO(2) and CH(3)COOH at room temperature for the formation of GaOOH and pseudomorphic and topotactic phase transformation from GaOOH to Ga(2)O(3). Growth of β-Ga 2O 3 nanocolumns crossing perpendicularly each other on MgO (1 0 0) surface Article Full-text available Mar 2005 J ALLOY COMPD Kyoko Hori Masahiro Fukuta Hirokazu Shimooka Yoshio Bando β-Ga2O3 nanocolumns straightened and crossed perpendicularly each other were deposited on MgO (100) substrate by vapor phase transport method. Growth of the nanocolumns was examined at steps of 1000, 1050, and 1200°C in elevation of source-boat temperature. We have drawn out the substrate from deposition-tube at each source-boat temperatures of 1000, 1050, and 1200°C. Scanning electron microscopy of the sample with source-boat temperature of 1200°C demonstrated that the straightened and elongated nanocolumns are crossing perpendicularly each other. Typical lengths of the nanocolumns were in the range of several hundreds nanometers below 1050°C, and those of 1200°C were in the range of ten to fifteen hundreds nanometers. Diameters of the nanocolumns stayed in the range of few hundreds nanometers, notwithstanding variation of the source temperature. X-ray diffraction and transmission electron microscopy with energy dispersive X-ray spectroscopy revealed that the nanocolumns are monoclinic β-Ga2O3 crystal, and the (400) plane of β-Ga2O3 nanocolumns is parallel to the (100) plane of MgO substrate. Catalytic Synthesis and Photoluminescence of ??-Ga2O3 Nanowires Article Full-text available May 2001 APPL PHYS LETT C. H. Liang G. W. Meng Guozhong Wang SY Zhang Monoclinic gallium oxide (β-Ga2O3) nanowires were synthesized by heat treating a composite material of GaAs and pre-evaporated Au at 1240 °C in dry oxygen atmosphere. The catalytic Au metal generated liquid nanoclusters that serve as reactive sites confining and directing the growth of β-Ga2O3 nanowires during the vapor-liquid-solid growth process. The β-Ga2O3 nanowires have diameters ranging from 20 to 50 nm and lengths of several micrometers. Photoluminescence measurement under excitation at 250 nm shows that the bulk β-Ga2O3 nanowires have a stable blue emission at 475 nm and an ultraviolet emission at 330 nm, which may be related to the defects such as the oxygen vacancy and the gallium–oxygen vacancy pair. © 2001 American Institute of Physics. Role of oxygen vacancy defect states in the n-type conduction of β-Ga2O3 Article Full-text available Oct 1999 Z. Hajnal Juan Carlos Miro Gábor Kiss J. Michael Kuperberg Based on semiempirical quantum-chemical calculations, the electronic band structure of beta-Ga2O3 is presented and the formation and properties of oxygen vacancies are analyzed. The equilibrium geometries and formation energies of neutral and doubly ionized vacancies were calculated. Using the calculated donor level positions of the vacancies, the high temperature n-type conduction is explained. The vacancy concentration is obtained by fitting to the experimental resistivity and electron mobility. (C) 1999 American Institute of Physics. [S0021-8979(99)03318-6]. Template-Free Synthesis of Highly Uniform α-GaOOH Spindles and Conversion to α-Ga2O3 and β-Ga2O3 Article Full-text available The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA. Biomimetic synthesis and patterning of silver nanoparticles Viral assembly of oriented quantum dot nanowires Pulmonary Toxicity of Single-Wall Carbon Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation We report a virus-based scaffold for the synthesis of single-crystal ZnS, CdS, and freestanding chemically ordered CoPt and FePt nanowires, with the means of modifying substrate specificity through standard biological methods. Peptides (selected through an evolutionary screening process) that exhibit control of composition, size, and phase during nanoparticle nucleation have been expressed on the highly ordered filamentous capsid of the M13 bacteriophage. The incorporation of specific, nucleating peptides into the generic scaffold of the M13 coat structure provides a viable template for the directed synthesis of semiconducting and magnetic materials. Removal of the viral template by means of annealing promoted oriented aggregation-based crystal growth, forming individual crystalline nanowires. The unique ability to interchange substrate-specific peptides into the linear self-assembled filamentous construct of the M13 virus introduces a material tunability that has not been seen in previous synthetic routes. Therefore, this system provides a genetic toolkit for growing and organizing nanowires from semiconducting and magnetic materials. Article Sep 2005 EUR J PHARM BIOPHARM The aim of this study was to explore potential use of the silk fibroin (SF) as an aqueous coating material for theophylline tablets. We have examined the film forming and coating properties of heat-treated fibroin, SF solution having different amounts of polyethylene glycol (PEG) and 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) cross-linked SF. Heat-treated SF material possessed a brittle structure, which resulted in poor film forming and coating properties. The optimum PEG amount in SF solution was determined as 17% (by weight) for an acceptable film forming and zero order release profile. EDC cross-linked SF has shown a very good film forming and coating property with a potential for sustaining the drug release from coated theophylline tablets. Dissolution data for coated theophylline tablets were analyzed using Ritger and Peppas equation to describe the mechanism of drug release. Drug release from the EDC coated tablets followed zero-order kinetics. Release rate constants were found to be 0.26, 0.19, 0.16%min(-1) for single-coated, double coated, and triple coated tablets, respectively. These results clearly demonstrated that silk fibroin has high utility as a novel aqueous coating material for controlled release products.	https://www.researchgate.net/publication/26302578_Biomineralization_of_Uniform_Gallium_Oxide_Rods_with_Cellular_Compatibility
Comfort and Joy: The Bittersweet Comedies of Bill Forsyth - Moving Image Source Melancholy is not an emotion that we usually associate with comedies, at least not in the laugh-a-minute, chortle-every-10-seconds type of movies that most audiences have come to expect from the genre.… Comfort and Joy: The Bittersweet Comedies of Bill Forsyth March 11–April 11, 2010 at Dryden Theatre at George Eastman House , Rochester, NYMelancholy is not an emotion that we usually associate with comedies, at least not in the laugh-a-minute, chortle-every-10-seconds type of movies that most audiences have come to expect from the genre. It takes a rare type of film artist to discover the almost inexpressible wistful sadness and small sense of loss behind the laughter and triumphs of comedic characters. Such an artist is the Scottish writer and director Bill Forsyth, whose sublime brand of filmmaking will be on display in the Dryden during March and April, when we present the first complete North American retrospective of his features. Forsyth will join us in person for screenings of his lovely movies, Local Heroand Housekeeping.Beginning his career in documentaries, Forsyth made his feature debut in 1980 with a low-budget comedy about a group of Glaswegian teenagers (played by members of Glasgow Youth Theatre) who relieve their boredom by stealing sinks and plumbing supplies. The four main actors in That Sinking Feeling(which wasn't released in the U.S. until 1984) were all cast in Forsyth's sophomore effort, Gregory's Girl, the story of a teenage boy's fixation on the first female member of his school's soccer team. These decidedly quirky first two features are youth comedies populated by unusually wise, even philosophical, youngsters, who make the bittersweet discovery that you can't always get what you want.The international success of Gregory's Girlpaved the way for Forsyth's next-and best-loved-movie, Local Hero. Though produced with support from Warner Bros. and starring two American actors (Peter Riegert and Burt Lancaster), Local Herois remembered today for its unique Scottish-ness and a subtle but ahead-of-its-time message on protecting our natural environment (it's Al Gore's favorite movie). Like Mac, the humbled oil executive hero of Local Hero, radio d.j. Dicky Bird in Comfort and Joyis another foiled romantic who finds himself embroiled in a misadventure; specifically, a war between Glasgow ice cream vendors.When the producer of Local Hero, David Putnam, was briefly named head of Columbia Pictures, he provided Forsyth the opportunity to make his first American movie. The result was the haunting and criminally neglected masterpiece Housekeeping, starring Christine Lahti as the eccentric guardian of two orphaned girls. Forsyth completed two more wonderful and underseen comedies in the U.S., Breaking In, starring Burt Reynolds and Casey Siemaszko as a professional burglar and his inexperienced protégé, and Being Human, featuring Robin Williams as five characters (or is it just one?) who learn through 10,000 years of history and heartbreak what it means to be alive.It's been more than a decade since Forsyth completed his last feature, Gregory's 2Girls, a sequel to one of his earlier successes that returned him to filming in Scotland. His body of work reminds us that there's a lot of comfort and joy and beautiful melancholy to be derived from life's ordinariness. Some might say there's no room for his subtle, quiet style of storytelling in a world dominated by increasingly bombastic popular culture, but seeing these films just might remind you that we need Bill Forsyth now more than ever.Featured Works: That Sinking Feeling(Bill Forsyth, 1979); Gregory's Girl(Bill Forsyth, 1981); Local Hero(Bill Forsyth, 1983,pictured); Comfort and Joy(Bill Forsyth, 1984); Housekeeping(Bill Forsyth, 1987); Breaking In(Bill Forsyth, 1989); Being Human(Bill Forsyth, 1994); Gregory's 2Girls(Bill Forsyth, 1999)Program information:Comfort and Joy: The Bittersweet Comedies of Bill ForsythRelated Articles:Lost BoysbyBilge Ebiriposted Apr. 15, 2010	http://www.movingimagesource.us/events/comfort-and-joy-the-bittersweet-comedies-20100311
(PDF) McRouter: Multicast within a router for high performance network-on-chips PDF \| The inevitable advent of the multi-core era has driven an increasing demand for low latency on-chip inter-connection networks (or NoCs). Being a... \| Find, read and cite all the research you need on ResearchGate Conference Paper PDF Available McRouter: Multicast within a router for high performance network-on-chips September 2013 Parallel Architectures and Compilation Techniques - Conference Proceedings, PACT DOI: 10.1109/PACT.2013.6618828 Conference: Parallel Architectures and Compilation Techniques (PACT), 2013 22nd International Conference on At: Edinburgh, Scotland Authors: <here is a image 518a9c36dd5288d3-d140d5aa4f2c1587> Yuan He Shenyang University of Technology <here is a image f2f62089ad6ff61c-d19572ff1996375a> Hiroshi Sasaki Hiroshi Sasaki <here is a image 19f2b21d74653845-bb1fe7ba5c132de5> Shinobu Miwa The University of Electro-Communications <here is a image f2f62089ad6ff61c-d19572ff1996375a> Hiroshi Nakamura Hiroshi Nakamura Abstract and Figures The inevitable advent of the multi-core era has driven an increasing demand for low latency on-chip inter-connection networks (or NoCs). Being a critical part of the memory hierarchy for modern chip multi-processors (CMPs), these networks face stringent design constraints to provide fast communication with tight power budget. Modern NoC's first-order concern is clearly its latency, while we also find that internal bandwidth of its routers is relatively plentiful; thus, we present a low latency router design utilizing a technique we call “multicast within a router” or McRouter, which allows productive utilization of remaining bandwidth inside a NoC router. McRouter allows a single cycle transfer of flits which shortens the communication latency when there is enough remaining bandwidth within the router. The key idea is to transmit a header flit to all possible output ports (multicast) so that it is always transmitted to the correct output port without relying on route computation. In addition, we find it is affordable with marginal power overhead while still being a stand-alone design by maintaining portability and modularity (unlike look-ahead routing based designs). Our evaluation with application traffic shows that McRouter helps achieving system speed-ups of 1.28, 1.17 and 1.05 over the conventional router (CR), the VSA router (VSAR) and the prediction router (PR), respectively. <here is a image 45002b2020a001bd-b0a0d4ab864a899c> Architecture of the conventional router. … <here is a image 6870220e4eeaa230-2d29354c57912100> 16-core CMP connected with NoC. … <here is a image 7f4e32fe1ac07426-98aeb5cb407183ec> Evaluations with synthetic traffic. … <here is a image 389aa05a607c5e55-9de079cfa8a371ec> System speed-up with router parameter downscaling. … ... Thirdly, routing at each hop and buffer allocations/accesses deepen the router pipelines and further increase the latency. To tackle these concerns, many optimization techniques are proposed [2,5,7,9,11, 13, [19][20][21][22]. Despite the effectiveness of such techniques directly addressing power and performance issues, there are also attempts that question the necessity of VC flow control, buffering and arbitrations in modern NoCs. ... ... On the other hand, there are many existing works focusing on shortening the latency of routers [9,12, 13, 16,[20][21][22]. Additionally, Kumar et al. proposed express channels which enable multi-hop packets to bypass intermediate routers [16]. ... Energy-Efficient On-Chip Networks through Profiled Hybrid Switching Conference Paper Full-text available <here is a image 518a9c36dd5288d3-d140d5aa4f2c1587> Yuan He Jinyu Jiao <here is a image 4b1968a375e9c6c8-7e8d7d788ed4c4e8> Thang Cao <here is a image 6f072a148b60daaf-7f208935db9238a0> Masaaki Kondo Virtual channel (VC) flow control is the de facto choice for modern networks-on-chip (NoCs) to allow better utilization of the link bandwidth through buffering and packet switching (PS), which are also the sources of large power footprint and long per-hop latency. However, bandwidth can be plentiful for parallel workloads under VC flow control. Thus, dated but simpler mechanisms, such as circuit switching (CS), can help improve the energy efficiency of modern NoCs. In this paper, we propose to apply CS to part of the link bandwidth so that a considerable amount of traffic can be transmitted bufferlessly without routing. Evaluations reveal that this proposal leads to a reduction of energy per flit by up to 32% while also provides very competitive latency when compared to networks under VC flow control. ... To tackle the above concerns, many optimization techniques are proposed [2,5,7,9,11, 13, [19][20][21][22]. Despite the effectiveness of such optimization techniques directly addressing power and performance issues of NoCs, there are also attempts which question the necessity of VC flow control and flit buffers in modern NoCs. ... ... On the other hand, there are many existing works focusing on shortening the latency of a router [9,12, 13, 16,[20][21][22]. Additionally, Kumar et al. proposed to have express channels which enable a multi-hop packet to bypass intermediate routers [16]. ... Energy-Efficient On-Chip Networks through Profiled Hybrid Switching Preprint <here is a image 518a9c36dd5288d3-d140d5aa4f2c1587> Yuan He Jinyu Jiao <here is a image 4b1968a375e9c6c8-7e8d7d788ed4c4e8> Thang Cao <here is a image 6f072a148b60daaf-7f208935db9238a0> Masaaki Kondo Virtual channel flow control is the de facto choice for modern networks-on-chip to allow better utilization of the link bandwidth through buffering and packet switching, which are also the sources of large power footprint and long per-hop latency. On the other hand, bandwidth can be plentiful for parallel workloads under virtual channel flow control. Thus, dated but simpler flow controls such as circuit switching can be utilized to improve the energy efficiency of modern networks-on-chip. In this paper, we propose to utilize part of the link bandwidth under circuit switching so that part of the traffic can be transmitted bufferlessly without routing. Our evaluations reveal that this proposal leads to a reduction of energy per flit by up to 32% while also provides very competitive latency per flit when compared to networks under virtual channel flow control. ... The work in [17] does VA and SA in parallel speculatively and prioritizes non-speculative packets in SA to increase resource utilization. The work in [18] exploits the abundant bandwidth inside the router and multicast flits to output ports speculatively rather than waiting for SA. Parallel processing of a packet can also happen on different routers with the help of control flits, which go ahead of data flits [19]. ... ... For the low-injection rate application Barnes, SlideAcross reduces the execution time by 38.1%, showing the effectiveness of bypassing. The SPLASH-2 workloads have low average network utilization [18] , so the performance under low-load conditions largely determines the system performance. ... Extending the Performance of Hybrid NoCs beyond the Limitations of Network Heterogeneity Article Full-text available <here is a image 43bedc4de833ab3b-68aa879e7989661d> Michael Opoku Agyeman <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Wen Zong <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Alex Yakovlev <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> T. Mak To meet the performance and scalability demands of the fast-paced technological growth towards exascale and big data processing with the performance bottleneck of conventional metal-based interconnects (wireline), alternative interconnect fabrics, such as inhomogeneous three-dimensional integrated network-on-chip (3D NoC) and hybrid wired-wireless network-on-chip (WiNoC), have emanated as a cost-effective solution for emerging system-on-chip (SoC) design. However, these interconnects trade off optimized performance for cost by restricting the number of area and power hungry 3D routers and wireless nodes. Moreover, the non-uniform distributed traffic in a chip multiprocessor (CMP) demands an on-chip communication infrastructure that can avoid congestion under high traffic conditions while possessing minimal pipeline delay at low-load conditions. To this end, in this paper, we propose a low-latency adaptive router with a low-complexity single-cycle bypassing mechanism to alleviate the performance degradation due to the slow 2D routers in such emerging hybrid NoCs. The proposed router transmits a flit using dimension-ordered routing (DoR) in the bypass datapath at low-loads. When the output port required for intra-dimension bypassing is not available, the packet is routed adaptively to avoid congestion. The router also has a simplified virtual channel allocation (VA) scheme that yields a non-speculative low-latency pipeline. By combining the low-complexity bypassing technique with adaptive routing, the proposed router is able to balance the traffic in hybrid NoCs to achieve low-latency communication under various traffic loads. Simulation shows that the proposed router can reduce applications’ execution time by an average of 16.9% compared to low-latency routers, such as SWIFT. By reducing the latency between 2D routers (or wired nodes) and 3D routers (or wireless nodes), the proposed router can improve the performance efficiency in terms of average packet delay by an average of 45 % (or 50 % ) in 3D NoCs (or WiNoCs). ... [7] does VA and SA in parallel speculatively, and prioritizes non-speculative packets in SA to increase resource utilization. [8] exploits the abundant bandwidth inside router and multicast flits to output ports speculatively rather than waiting for SA. Parallel processing of a packet can also happen on different routers with the help of control flits which goes ahead of data flits [9]. ... ... where ψ ≤ min (8) where new R and old R are the proposed new router and conventional 2D routers micro-architecture, respectively. A x and P x represents the area and power consumption of router x. ... An Efficient 2D Router Architecture for Extending the Performance of Inhomogeneous 3D NoC-Based Multi-Core Architectures Conference Paper <here is a image 43bedc4de833ab3b-68aa879e7989661d> Michael Opoku Agyeman ... With such a background, there are many existing studies on optimizing performance and power consumption of NoCs. Low latency routers are used to reduce transmission latency [2]- [6] . Hybrid router buffer designs attempt to improve the network throughput [7]. ... ... Not surprisingly, many existing NoC optimization techniques have already been studied in order to improve the performance or power of NoCs. Performance-wise, there are many existing router designs focusing on shortening the latency of a router [2]- [6] , [12], [13]. There are also studies which focus on improving the bandwidth of NoCs through different approaches, such as compression [9], [10], allocators with higher matching efficiency [8], and different buffer designs allowing higher capacity [7]. ... A Runtime Optimization Selection Framework to Realize Energy Efficient Networks-on-Chip Article Full-text available IEICE T INF SYST <here is a image 518a9c36dd5288d3-d140d5aa4f2c1587> Yuan He <here is a image 6f072a148b60daaf-7f208935db9238a0> Masaaki Kondo Takashi Nakada Hiroshi Nakamura Networks-on-Chip (or NoCs, for short) play important roles in modern and future multi-core processors as they are highly related to both performance and power consumption of the entire chip. Up to date, many optimization techniques have been developed to improve NoC’s bandwidth, latency and power consumption. But a clear answer to how energy efficiency is affected with these optimization techniques is yet to be found since each of these optimization techniques comes with its own benefits and overheads while there are also too many of them. Thus, here comes the problem of when and how such optimization techniques should be applied. In order to solve this problem, we build a runtime framework to throttle these optimization techniques based on concise performance and energy models. With the help of this framework, we can successfully establish adaptive selections over multiple optimization techniques to further improve performance or energy efficiency of the network at runtime. ... Many works focusing on shortening the network latency through more advanced router designs at the cost of power consumption exist [5], [8] , [10]- [13]. Moreover, many optimization techniques were also proposed to help cut the power consumption of NoCs. ... Local Traffic-Based Energy-Efficient Hybrid Switching for On-Chip Networks Conference Paper <here is a image 518a9c36dd5288d3-d140d5aa4f2c1587> Yuan He Jinyu Jiao <here is a image 6f072a148b60daaf-7f208935db9238a0> Masaaki Kondo Advanced flow control mechanisms employed by modern on-chip networks are the reasons of large energy footprint and long per-hop latency. On the other hand, dated and simpler flow controls such as circuit switching can draw far less power and offer an end-to-end latency analogous to wire delay. In this paper, we present a hybrid flow control mechanism, which mixes both virtual channels and circuit-switching, to provide a latency-competitive and energy-efficient on-chip network design. Contrary to existing hybrid switching designs, our proposal is based on local traffic so that circuits are formed without the knowledge of end-to-end traffic. When compared to on-chip networks with virtual channels, our proposal achieves a very competitive latency per flit, for up to 4% lower, while also dramatically suppressing the energy per flit by up to 18%. ... [7] does VA and SA in parallel speculatively, and prioritizes non-speculative packets in SA to increase resource utilization. [8] exploits the abundant bandwidth inside router and multicast flits to output ports speculatively rather than waiting for SA. Parallel processing of a packet can also happen on different routers with the help of control flits which goes ahead of data flits [9]. ... An efficient 2D router architecture for extending the performance of inhomogeneous 3D NoC-based multi-core architectures Conference Paper Full-text available <here is a image 43bedc4de833ab3b-68aa879e7989661d> Michael Opoku Agyeman ... Here non-speculative packets are prioritized in SA to increase resource utilization. [10] exploits the abundant bandwidth in routers and multicast flits to output ports speculatively rather than wait for SA. Parallel processing of a packet can also be implemented on different routers with the help of control flits which goes ahead of data flits [15]. ... On Improving the Performance of Hybrid Wired-Wireless Network-on-Chip Architectures Conference Paper <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Wen Zong <here is a image 43bedc4de833ab3b-68aa879e7989661d> Michael Opoku Agyeman Recently, hybrid wired-wireless Network-on-Chip (WiNoC) have been proposed to meet the performance and scalability demands of modern System-on-Chip (SoC) design. However, due to the presence of wirelines with multi-hop nodes in the hybrid architecture, WiNoCs have reduced performance efficiency. In this paper, we propose a low-complexity single-cycle bypassing mechanism to alleviate the performance degradation in such emerging hybrid NoCs. The proposed router employs both dimension-ordered routing (DoR) and a deadlock free adaptive routing to transmit flits at low-loads and high traffic loads, respectively, to efficiently balance traffic in WiNoCs. By reducing the latency between the wired nodes and the wireless nodes, the proposed router can improve performance efficiency in terms of average packet delay by an average of 50% in WiNoCs. Traffic-Aware Energy-Efficient Hybrid Input Buffer Design for On-Chip Routers Conference Paper	https://www.researchgate.net/publication/261523446_McRouter_Multicast_within_a_router_for_high_performance_network-on-chips
SARS-CoV-2 induces human endogenous retrovirus type W envelope protein expression in blood lymphocytes and in tissues of COVID-19 patients \| medRxiv medRxiv - The Preprint Server for Health Sciences SARS-CoV-2 induces human endogenous retrovirus type W envelope protein expression in blood lymphocytes and in tissues of COVID-19 patients View ORCID Profile Benjamin Charvet , View ORCID Profile Joanna Brunel , Justine Pierquin , Mathieu Iampietro , View ORCID Profile Didier Decimo , Nelly Queruel , View ORCID Profile Alexandre Lucas , María del Mar Encabo-Berzosa , Izaskun Arenaz , Tania Perez Marmolejo , Francina Valezka Bolaños Morales , Arturo Ivan Gonzalez Gonzalez , View ORCID Profile Armando Castorena Maldonado , César Luna Rivero , View ORCID Profile Cyrille Mathieu , Patrick Küry , Jose Flores-Rivera , View ORCID Profile Santiago Avila Rios , View ORCID Profile Gonzalo Salgado Montes de Oca , View ORCID Profile Jon Schoorlemmer , View ORCID Profile Branka Horvat , View ORCID Profile Hervé Perron doi: https://doi.org/10.1101/2022.01.18.21266111 Abstract Abstract Patients with COVID-19 may develop abnormal inflammatory response and lymphopenia, followed in some cases by delayed-onset syndromes, often long-lasting after the initial SARS-CoV-2 infection. As viral infections may activate human endogenous retroviral elements (HERV), we studied the effect of SARS-CoV-2 on HERV-W and HERV-K envelope (ENV) expression, known to be involved in immunological and neurological pathogenesis of human diseases. Our results have showed that the exposure to SARS-CoV-2 virus activates early HERV-W and K transcription but only HERV-W ENV protein expression, in an infection- and ACE2-independent way within peripheral blood mononuclear cell cultures from one-third of healthy donors. Moreover, HERV-W ENV protein was significantly increased in serum and plasma of COVID-19 patients, correlating with its expression in CD3 +lymphocytes and with disease severity. Finally, HERV-W ENV was found expressed in post-mortem tissues of lungs, heart, brain olfactory bulb and nasal mucosa from acute COVID-19 patients in cell-types relevant for COVID-19-associated pathogenesis within affected organs, but different from those expressing of SARS-CoV-2 antigens. Altogether, the present study revealed that SARS-CoV-2 can induce HERV-W ENV expression in cells from individuals with symptomatic and severe COVID-19. Our data suggest that HERV-W ENV is likely to be involved in pathogenic features underlying symptoms of acute and post-acute COVID. It highlights the importance to further understand patients‘ genetic susceptibility to HERV-W activation and the relevance of this pathogenic element as a prognostic marker and a therapeutic target in COVID-19 associated syndromes. Competing Interest Statement HP, BC, JB, JP, NQ received compensation for their work from Geneuro Innovation (France) Funding Statement This study was funded by the french National Agency for Researsch (ANR) - program RA-COVID-19 V15 (project name : COVERI) Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: - Ethics committee/IRB of SECRETARIA DE SALUD INSTITUTO NACIONAL DE ENFERMEDADES RESPIRATORIAS ISMAEL COSIO VILLEGAS (MEXICO)gave ethical approval for this work - Ethics committee/IRB of HOSPICES CIVILS DE LYON (HCL)/ BIOBANK OF CROIX ROUSSE HOSPITAL (FRANCE) gave ethical approval for this work - Ethics committee/IRB of SERVICIOS CIENTIFICO TECNICOS - SERVICIO DE BIOBANCO (BSSA)OF UNIVERSITY OF SARAGOZA (SPAIN) gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Paper in collection COVID-19 SARS-CoV-2 preprints from medRxiv and bioRxiv medRxiv 2022.01.18.21266111; doi: https://doi.org/10.1101/2022.01.18.21266111 COVID-19 SARS-CoV-2 preprints from medRxiv and bioRxiv Subject Area Infectious Diseases (except HIV/AIDS) doi: https://doi.org/10.1101/2022.01.18.21266111	https://www.medrxiv.org/content/10.1101/2022.01.18.21266111v1
Farm-level medicinal plants production and economic analysis in the Indian state of Punjab \| Request PDF Request PDF \| Farm-level medicinal plants production and economic analysis in the Indian state of Punjab \| Farmers all across the world conduct experiments, and have done so for a long time, way before modern experimental science and its forerunners... \| Find, read and cite all the research you need on ResearchGate Farm-level medicinal plants production and economic analysis in the Indian state of Punjab April 2023 Journal of Applied Research on Medicinal and Aromatic Plants 34(2):100486 DOI: 10.1016/j.jarmap.2023.100486 Neha Bajwa Lakhwinder Sharma Maharaja Ranjit Singh Punjab Technical University, Bathinda, Punjab, India Request full-text Abstract Farmers all across the world conduct experiments, and have done so for a long time, way before modern experimental science and its forerunners were acknowledged. Farmer techniques, based on their experience, have some advantages over agricultural specialists' field trials under the relevant local conditions, making documentation of successful agricultural practises essential. In this article, we document the successful farmer’s practices of selected medicinal plants i.e. Aloe vera, Ocimum sanctum, and Curcuma longa in the Indian state of Punjab through farm-level observations and data. Following that, the cost-return structure of these medicinal plants was examined using average prices obtained from personal interviews utilising a questionnaire in order to demonstrate their economic potential over traditional crops (wheat and paddy). The study highlights that Aloe vera has Indian Rupee (INR) 92,876 (1127.55 USD) profit per one acre area, Curcuma longa has INR. 73,725 (895.05 USD) profit per one acre area, while Ocimum sanctum have INR 35,515 (431.16 USD) return over variable cost which is comparatively higher than wheat and paddy. The article underlines Ocimum sanctum as a successful competitor and replacer of paddy in Kharif/Monsoon season. The study's selection of common agricultural indicators and agricultural practises was mostly dependent on the inputs and average values provided by the farmers. Little variations, however, that cannot be measured to arrive at the compiled data may exist. The economic analysis in the present study is representable to only Punjab, however, the cost-return structure may vary in different states, and nations depending on ecological, and trade patterns. This article will serve as a guidance document for farmers interested in medicinal plant adoption owing to its on-field effective farmer practises and agro-economics. Highlighting Agro-ecological Zones to Upscale the Production of Immunity-booster Plants in Punjab Article Full-text available Jun 2021 Preet Amol Singh Anil Sood Ashish Baldi Witnessing an increase in the demand for immunity-booster plants especially in the aftermath of COVID-19, it becomes imperative to highlight suitable growing areas for Withania somnifera (L.) Dunal (Ashwagandha), and Ocimum sanctum L. (Tulsi) in Punjab. The scope of this work is to suggest market-driven immunity-booster plants that can act as a substitute for high water, pesticides, and fertilizers-dependent rice crop to promote sustainable agriculture. The agro-ecological zoning model was devised by highlighting agro-climatic aptitude requirements of the plants and mapping their requirements with the agro-ecological conditions of Punjab with special emphasis on temperature, rainfall, soil texture, and pH to highlight optimally suitable, suitable, and lesser suitable zones for the plants. The suitability maps were prepared using Geographic Information System. Ashwagandha was optimally suitable for zones-II, III; suitable for zones-I IV; and lesser suitable for zone-V. Similarly, Tulsi was optimally suitable for zones-I, II, III and suitable for zones-IV and V. With no policy to regulate the right medicinal plants in the right area, this agro-ecological study is essential to upscale the production of immunity-booster plants. A Comparative Review on the Standard Quality Parameters of Turmeric Article Full-text available Mar 2021 Preet Amol Singh Neha Bajwa Ashish Baldi Farmers’ experiments and scientific methodology Article Full-text available Oct 2019 Sven Ove Hansson Farmers all over the world perform experiments, and have done so since long before modern experimental science and its recognized forerunners. There is a rich anthropological literature on these experiments, but the philosophical issues that they give rise to have not received much attention. Based on the anthropological literature, this study investigates methodological and philosophical issues pertaining to farmers’ experiments, including the choice of interventions (work methods etc.) to be tested, the planning of experiments, and the use of control fields and other means to deal with confounding factors. Farmers’ experiments have some advantages over the field trials of agricultural scientists (more replications, studies performed under the relevant local conditions), but also some comparative disadvantages (less stringent controls, less precise evaluations). The two experimental traditions are complementary, and neither of them can replace the other. Several aspects of farmers’ experiments are shown to have a direct bearing on central topics in the philosophy of science. View Good Agricultural Practices for Aloe Good Agricultural Practices for Aloe Compiled by Mar 2015 Sanskar SINGH Jat R Nagaraja Reddy Ruchi Bansal P Manivel A Review on Plant Antimicrobials of Past Decade Full-text available May 2018 CURR TOP MED CHEM Preet Amol Singh Sapna Desai Jasbir Singh As per WHO reports, about three-quarters (65-80%) of the world's population seeks plants or plant-derived natural products for various diseases. The slow discovery of new synthetic molecules and rising resistance in microbes against existing ones has triggered an alarm for speeding up the development process for new molecules. Traditional system(s) of medicine and plant resources has been foresighted again by researchers to circumvent the situation. This review represents various plant genera which, either as a whole plant or their parts, have been reported possessing antimicrobial properties during the last decade. Before 2007, literature is already well cited in various books and reviews. Economics of Production to Marketing of Aromatic Crops in Uttar Pradesh: A Case Study § Article Full-text available Jun 2012 Ram Suresh Sharma Sanjay Kumar Virendra Singh Anil Kumar Singh The economics of production of three medicinal and aromatic plants, viz. menthol mint, tulsi and vetiver has been worked out using farm-level data from the districts of Barabanki, Sitapur and Raebaerli in Uttar Pradesh. The cultivation of these plant species has been found to be highly profitable, and farmers of these districts need to be made aware about this fact. The net returns over total cost have been found higher for vetiver (` 1, 53,933 /ha), followed by menthol mint (` 53,250 / ha) and tulsi (`40,094 /ha). The benefit– cost ratio however has been observed to be highest for menthol mint (3.27), followed by tulsi (3.21) and vetiver (3.04). The employment generation potential of these three crops has also been found quite high. The education of farmers has been found to be influencing the cultivation of aromatic crops. The study has drawn attention to the following aspects: (i) development of a package of improved cultivation practices, (ii) availability of good quality planting material of high-yielding, short-duration varieties, (iii) more demonstration / extension efforts, (iv) better access to timely and adequate credit, (v) access to timely market information, (vi) establishment of regulated market, (vii) introduction of minimum support price. (viii) contract farming. (ix) establishment of testing facilities, and (x) linkage with pharmaceutical and perfumery industries so as to boost cultivation of aromatic crops in the study area. Integrated weed management in poplar (Populus deltoides)–turmeric (Curcuma longa) based agroforestry system in Punjab Article Full-text available Jul 2016 Navneet Kaur Rishi I S Gill Makhan Singh Bhullar A field study was conducted during 2013–14 and 2014–15 to find out the effective and economical approach for weed control in turmeric (Curcuma longa L.) grown under poplar (Populus deltoides Bartr.) block plantation which was established at 4 m × 3 m spacing in January 2012. Three pre-emergence herbicides (pendimethalin 0.75 and 1.0 kg/ha, oxyfluorfen 0.176 and 0.235 kg/ha, atrazine 0.563 and 0.75 kg/ha) were integrated with 9 t/ha paddy straw mulching during both the years except with atrazine during 2014–15. Integrated use of oxyfluorfen 0.235 kg/ha or pendimethalin 1.0 kg/ha with straw mulch provided similar level of weed control to that of 3 hand-weedings and increased rhizome yield of turmeric by 36–42% over weedy check. Pendimethalin 1.0 kg/ha + straw mulch recorded higher benefit: cost ratio than hand-weedings. Atrazine + straw mulch recorded effective control of weeds, however it killed the poplar trees at both the levels; hence not safe for use in turmeric grown in poplar plantations. Treatment combinations of pendimethalin and oxyfluorfen with straw mulch were found to be safe for weed management in poplar-turmeric based agroforestry system. Influence of mulching and irrigation scheduling on productivity and water use of turmeric (Curcuma longa L.) in north-western India Article Full-text available Jul 2016 IRRIGATION SCI Amandeep Kaur A. S. Brar A field experiment was conducted to compute the water use and productivity of turmeric as a function of straw mulching and irrigation scheduling at Punjab Agricultural University, Ludhiana, during 2013 and 2014. The experiment was laid out in split plot design, keeping mulch levels (no mulch and straw mulch 6 t/ha) and irrigation methods (drip and check basin) in main plots and irrigation schedules at 0.6, 0.8, 1.0 and 1.2 irrigation water/cumulative pan evaporation (IW/CPE) in subplots. Turmeric yield was 125.2 % higher with mulching than no mulch with 50 % saving in irrigation water. Drip irrigation resulted in significantly higher turmeric yield and benefit/cost (B/C) than check basin. Irrigation scheduling at 1.2 IW/CPE recorded significantly higher turmeric yield than other schedules. Drip irrigation at 0.8 IW/CPE resulted in statistically at par yield with check basin irrigation at 1.2 IW/CPE, thus saving 40 % irrigation water with significantly higher B/C. However, turmeric yield was at par between drip irrigation at 1.2 and 1.0 IW/CPE schedule, while a significant reduction in yield was recorded in check basin at 1.0 IW/CPE compared to 1.2 IW/CPE. Turmeric should be irrigated with drip at 1.0 and with check basin at 1.2 IW/CPE to realize potential yield. Elucidating hydrochemical properties of groundwater for drinking and agriculture in parts of Punjab, India Article Full-text available Mar 2016 ENVIRON EARTH SCI Tenzin Thakur Madhuri S Rishi Pradeep K. Naik Prerna Sharma Groundwater over-exploitation leading to quality deterioration is an accepted fact, but limited work in this direction has been carried out in the Indian state of Punjab, where groundwater development is 172 %, the highest of any state in India. This paper makes an assessment of the groundwater quality in three administrative blocks of Rupnagar district in Punjab, in two of which groundwater exploitation has already reached about 200 %. Sixty groundwater samples were collected from shallow tubewells and hand pumps in premonsoon season (May, 2013) and were analyzed for major chemical parameters. Analytical results do not show any perceptible deterioration in groundwater quality; all the parameters are well within the permissible limits of the Indian standards, except for electrical conductivity (EC), total dissolved solids (TDS), Ca and Mg, which exceed the desirable limits in few groundwater samples. Abundance of ions in the water samples is in the order: HCO3− > Cl− > SO42− > NO3− and Mg2+ > Ca2+ >N a+ > K+. Data plotted on the US Salinity Laboratory diagram show that most of the samples fall in the category of C2S1 and C3S1, which makes the groundwater suitable for irrigation in all types of soil. Other chemical indices such as % sodium (%Na), sodium absorption ratio (SAR), and residual sodium carbonate (RSC) also show that almost all samples are fit for irrigation purposes. This work thus concludes that groundwater quality deterioration due to irrigation has not yet been well perceived in the state of Punjab, even if much of its groundwater resources have been over-exploited. However, the paper warns against indiscriminate drilling and over-exploitation of the groundwater resources for their long-term usage and sustainability. Agronomic practices for the production of Safed musli (Chlorophytum borivilianum Santapau & Fernandes) in India Article Full-text available Aparbal Singh Suman P S Khanuja Saudan Singh Medicinal and Aromatic Plants for Enhancing Farm Income: The Case of Bihar Article Full-text available Oct 2013 K. M. Singh Abhay Kumar R.K.P. Singh Ujjwal Kumar The MAPs provide opportunities for developing a variety of safe and cost effective, prophylactic, and curative medicines for a number of maladies. It is estimated that the primary health care of over 80 per cent of the world's population still depends on plant based traditional medicines (WHO, 2002). Growing consciousness about health and side effects of modern medicines has again set the stage for innovation and use of herbal medicines. Evidence shows that the total domestic potential for crude drugs and oil extracts in India is worth Rs 3 billion, of which the requirements of over-the-counter products. Realizing the vast untapped potentials of MAPs and impediments in their development. ATMA has follwoed a systematic approach while taking MAPs for pilot testing. It made assessement about the local conditions and requirements, and demand for MAPs; strengthened capacity by traing various stakeholders; played role in supporting individuals and institutions; developed relevant literature in local language; emphasized on group approach by organizing the growers; encouraged cultivation through the organized sector with linking the unorganized farmers; and evolved a pricing mechanism with buy back arrangement with partner industry under public-private partnership(PPP). The present study tries to outline some of the steps taken to promote MAPs in Bihar for better income to the farmers. Effect of method of planting and harvesting time on growth, yield and quality of turmeric (Curcuma longa L.) Article Full-text available Apr 2009 Balwinder Kumar B S Gill Abstract An experiment was conducted at Ludhiana (Punjab) to study the influence of method of planting (flat and ridge planting) and harvesting time (10 November-12 March) on growth, yield and of quality turmeric (Curcuma longa). Planting methods did not influence growth, yield and quality of turmeric significantly. Harvesting on 12th March produced maximum fresh rhizome yield of 28.94 t ha-1 (mean yield) of turmeric which was statistically on par with 20th February (27.61 t ha-1) and 30th January (26.78 t ha-1) harvesting, but was significantly better than all the earlier harvesting dates. A similar trend was observed in processed turmeric yield. The number and weight of rhizomes improved significantly with delay in harvesting. The oil and curcumin content also increased with delay in harvesting. Enrichment, Development, and Assessment of Indian Basil Oil Based Antiseptic Cream Formulation Utilizing Hydrophilic-Lipophilic Balance Approach Article Full-text available Jan 2013 BMRI Narayan Prasad Yadav Jay Gopal Meher Neelam Pandey Debabrata Chanda The present work was aimed to develop an antiseptic cream formulation of Indian basil oil utilizing hydrophilic-lipophilic balance approach. In order to determine the required-hydrophilic lipophilic balance (rHLB) of basil oil, emulsions of basil oil were prepared by phase inversion temperature technique using water, Tween 80, and Span 80. Formulated emulsions were assessed for creaming (BE9; 9.8, BE10; 10.2), droplet size (BE18; 3.22 ± 0.09 μ m), and turbidity (BE18; 86.12 ± 2.1%). To ensure correctness of the applied methodology, rHLB of light liquid paraffin was also determined. After rHLB determination, basil oil creams were prepared with two different combinations of surfactants, namely, GMS : Tween 80 (1 : 3.45) and SLS : GMS (1 : 3.68), and evaluated for in vitro antimicrobial activity, skin irritation test, viscosity and consistency. The rHLB of basil oil and light liquid paraffin were found to be 13.36 ± 0.36 and 11.5 ± 0.35, respectively. Viscosity, and consistency parameters of cream was found to be consistent over 90 days. Cream formulations showed net zone of growth inhibition in the range of 5.0-11.3 mm against bacteria and 4.3-7.6 mm against fungi. Primary irritation index was found to be between 0.38 and1.05. Conclusively stable, consistent, non-irritant, enriched antiseptic basil oil cream formulations were developed utilizing HLB approach. Trichoderma as a Seed Treatment to Control Helminthosporium Leaf Spot Disease of Chrysalidocarpus lutescens Article Full-text available Jan 2009 Jegathambigai Velmurugu Shanthi Wilson Wijeratnam R. L. C. Wijesundera 3 Abstract: Helminthosporium (Bipolaris) causes a leaf spot disease in Chrysalidocarpus lutescens (cane palm) in Sri Lanka. The losses could reach 90% of production during rainy weather conditions. Field experiments were carried out to test the efficacy of treatment of seeds of C. lutescens against Helminthosporium infection. In the fungal growth tests the isolates T.harzianum 1, T.harzianum 2, T.viride 1,T.viride 2 and T.viride 3 inhibited growth of the pathogen by 79.18, 69.03, 83.75, 82.99, 74.11% respectively. Isolates of Trichoderma harzianum and Trichoderma viride obtained from soil and having antagonistic activity against Helminthosporium were used in the field experiments. In the field trials, seed treatment with a spore suspension of Trichoderma completely eliminated the disease while Trichoderma and Pseudomonas fluorescens reduced the disease incidence significantly. The seed treatments also significantly increased seed germination, seedling growth and seedling vigor. Effectiveness of Minimum Support Price Policy for Paddy in India with a Case Study of Punjab Article Full-text available Jan 2012 Shayequa Zeenat Ali R.s. Sidhu Kamal Vatta The effectiveness of minimum support price (MSP) for paddy has been examined in different regions of India and its role and contribution towards production in surplus states like Punjab have been studied. Based on the secondary data spanning from 1980-81 to 2006-07, the deviations of farm harvest prices from the MSP have been used as a measure of ineffectiveness and the impact of prices and technology on rice productivity has been examined by using the simultaneous equation model. While the MSP policy has been very effective in surplus producing states like Punjab and Andhra Pradesh, it has not been so effective in the deficit states. In Punjab, the effective implementation of the price policy has helped in improving the production and productivity of rice. Non-price factors such as use of improved varieties, availability of assured irrigation at subsidized rates and high fertilizer-use have been found to be significant determinants of growth in rice production. The study has suggested that without losing sight of the environmental concerns, the Punjab model can be used for increasing the production of rice in other potential areas of the country. Revitalizing Indian system of herbal medicine by the National Medicinal Plants Board through institutional networking and capacity building Article Full-text available Sep 2007 CURR SCI INDIA Kala Chandra Prakash Kala Bikram Singh Sajwan In view of the leading role of the National Medicinal Plants Board (NMPB) in India for developing the medicinal plants sector, the present study was carried out for exploring the functions and major initiatives taken up by NMPB for developing the medicinal plants sector. NMPB has successfully set up 35 State Medicinal Plants Boards for dealing with various aspects of medicinal plants at the state level. From April 2001 to March 2006, NMPB has sanctioned 4254 projects under two major schemes, viz. promotional and contractual farming. Besides funding projects, NMPB is engaged in a number of activities, including development of monographs on good agricultural and collection practices, an e-portal on market information, supporting projects for raising quality planting material and setting up of herbal gardens in schools. The major strategic role of NMPB is to generate livelihood options and employment as well as conservation of medicinal plants. The information presented in this article will help disseminate information for those seeking assistance and willing to know about the initiatives taken at the national level by the Government of India for developing the medicinal plants sector. Natural products derived from plants as a source of drugs Full-text available Oct 2012 Ciddi Veeresham Medicinal Plants, Conservation and Livelihoods Article Full-text available Jan 2004 Alan Charles Hamilton Many types of action can be taken in favour of the conservation and sustainable use of medicinal plants. Some of these are undertaken directly at the places where the plants are found, while others are less direct, such as some of those relating to commercial systems, ex situ conservation and bioprospecting. In the latter cases, actions taken will not lead to in situ conservation unless they feedback to improvements in the field. Probably the single most important role for medicinal plants in biological conservation is their use to achieve conservation of natural habitats more generally. This stems from the special meanings that medicinal plants have to people, related to the major contributions that they make to many people's lives in terms of health support, financial income, cultural identity and livelihood security. Problems associated with biopiracy or (the other side of the coin) excessive restrictions on research have come to assume policy prominence in the general thematic area of medicinal plant conservation and use. The fair and equitable sharing of benefits from bioprospecting is required under the Convention on Biological Diversity, but it is not always easy to achieve these ideals in practice. While experience is accumulated in how this may practically be achieved, it is important, at the present time, that controls imposed on scientific research to prevent biopiracy or theft of local and indigenous intellectual property do not unduly restrict research that has little or nothing to do with these matters. Current Status of Medicinal Plants Used by Traditional Vaidyas in Uttaranchal State of India Article Full-text available Dec 2005 Chandra Prakash Kala The current status of medicinal plants used by traditional Vaidyas was studied in Uttaranchal state of India. Information was gathered using semi-structured questionnaires among 60 traditional Vaidyas. They were questioned about the types of ailments treated with plants and the preparation of herbal medical formulations. A total of 243 herbal medical formulations prepared by Vaidyas treating 73 different ailments were documented. Plants were the major ingredients in these medical formulations. 156 medicinal plant species were documented during the survey. Of these 55% were cultivated and 45% were wild species. Of the cultivated species 80% were found growing in the kitchen gardens and 20% in the agricultural fields. The frequency of use of kitchen garden species was highest in preparing the medical formulations as in 243 formulations the relative frequency of use of such species was 87%. The relative frequency of use of the medicinal plants growing in the wild was 55% in preparing herbal medical formulations. There was a sharp decline in the number of traditional Vaidyas through generations. The loss of knowledge on preparing medicine was due to several reasons including the number of Vaidyas coming forward to adopt this traditional healing practice professionally. Pharmacovigilance of herbal medicines: Concerns and future prospects Article Mar 2023 J ETHNOPHARMACOL Abinash Choudhury Preet Amol Singh Neha Bajwa Preeti Bisht Abstract Ethnopharmacological relevance The use of herbal medicines for prophylaxis, prevention, and treatment of various ailments is rising throughout the world because they are thought to be safer than allopathic treatments, which they are. However, several investigations have documented the toxicity and adverse drug reactions (ADR) of certain formulations and botanicals if not consumed wisely. Aim of the study The goal of the current study is to address herbal medication pharmacovigilance (PV) modeling and related considerations for improved patient safety. Also, focus is laid on the comprehensive and critical analysis of the current state of PV for herbal medications at the national and international levels. Materials and methods Targeted review also known as focused literature review methodology was utilized for exploring the data from various scientific platforms such as Science Direct, Wiley Online Library, Springer, PubMed, Google Scholar using “pharmacovigilance, herbal medicine, traditional medicine, ADR, under reporting, herb toxicity, herb interactions” as keywords along with standard literature pertaining to herbal medicines that is published by the WHO and other international and national organizations etc. The botanical names mentioned in the present article were authenticated using World Flora Online database. Results: The historical developments paving the way for PV in regulatory setup were also discussed, along with various criteria's for monitoring herbal medicine, ADR of herbs, phytoconstituents, and traditional medicines, herb-drug interactions, modes of reporting ADR, databases for reporting ADR's, provisions of PV in regulatory framework of different nations, challenges and way forward in PV are discussed in detail advocating a robust drug safety ecosystem for herbal medicines. Conclusion Despite recent efforts to encourage the reporting of suspected ADRs linked to herbal medicines, such as expanding the programme and adding community pharmacists as recognized reporters, the number of herbal ADR reports received by the regulatory bodies remains comparatively low. Since users often do not seek professional advice or report if they have side effects, under-reporting, is anticipated to be significant for herbal medications. There are inadequate quality control methods, poor regulatory oversight considering herbs used in food and botanicals, and unregulated distribution channels. In addition, botanical identity, traceability of herbs, ecological concerns, over-the-counter (OTC) herbal medicines, patient-physicians barriers requires special focus by the regulatory bodies for improved global safety of herbal medicines. Determining constraints in medicinal plants adoption: A model geospatial study in the Indian state of Punjab Aug 2021 Preet Amol Singh Anil Sood Ashish Baldi Understanding the constraints involved in the adoption of medicinal plant cultivation is the most important step to design future policies and action plans for effective outcomes. Keeping this in view, a model survey-based study involving medicinal plant cultivators by using a purposing sampling technique was carried out. Additionally, farmers identified via exploration of various channels from public and personal sources were geo-tagged using Geographic Information System (GIS). The digital maps shall act as a platform to establish linkage between concerned stakeholders viz. policymakers, traders, industries, regulatory agencies, and farmers. The scope of the study was limited to progressive farmers cultivating commercially important medicinal plants i.e. Curcuma longa L. (Haldi), Aloe vera (Linn.) Burm.f (Ghritkumari), Phyllanthus emblica L. (Amla), Ocimum sanctum L. (Tulsi), Rauvolfia serpentina (L.) Benth. ex Kurz (Sarpgandha). The survey questionnaire was developed through the expert advisory method, followed by statistical validation based on achieving Chronbach’s alpha value of 0.6. Statistical analysis of the responses by applying Mean Percentage Score (MPS) and significance determination using Chi-square test indicated prominent correlation among small and large farmers in context to technical, trade, social, and awareness aspects. The study highlighted the non-availability of quality planting material, lack of awareness about good agricultural and packaging practices, less knowledge about agro-climatic suitability, and commercialization avenues acted as one of the major constraints. The study will act as a basis to frame policies for promoting medicinal plant cultivation as a commercially successful crop diversification model in the region. Possible role of traditional systems of medicine to manage covid-19: A review Article Jan 2021 ISR J PLANT SCI Preet Amol Singh Neha Bajwa Ashish Baldi The covid -19 pandemic has wreaked havoc on the global healthcare system and the economy. At present, no specific antiviral vaccine is available to combat this contagious virus. Traditional medicine has a long history of playing a significant role in managing several infectious diseases. In this context, scientists around the globe are also exploring various traditional medical interventions to prevent the covid -19 pandemic. In the present work, we summarize available scientific data advocating the use of traditional medicine for preventing covid -19. A robust literature review was conducted using scientific platforms such as Science Direct, National Center for Biotechnology Information ( ncbi ), Pubmed, Google Scholar, and online database like The Plant List (The Plant List 2013) version 1.1. Special emphasis was given to potential natural antiviral, immuno-modulator plants, and traditional medicines to highlight their possible roles in reducing the disease burden. Immuno-modulator such as Withania somnifera and other natural compounds especially glycyrrhizin, kaempferol, ginsenoside, and lycorine can be leading candidates against sars -CoV-2. Besides the need for rigorous scientific validation of potential herbs and related formulations, their use can be beneficial for the preventive as well as symptomatic treatment of covid -19 infected patients. This work provides a run-through of the experimental therapeutics, preventive and treatment options for covid -19. Purposive sampling: complex or simple? Research case examples Jun 2020 Sarah Prior Steve Campbell Melanie Greenwood Sarah Young Background Purposive sampling has a long developmental history and there are as many views that it is simple and straightforward as there are about its complexity. The reason for purposive sampling is the better matching of the sample to the aims and objectives of the research, thus improving the rigour of the study and trustworthiness of the data and results. Four aspects to this concept have previously been described: credibility, transferability, dependability and confirmability. Aims The aim of this paper is to outline the nature and intent of purposive sampling, presenting three different case studies as examples of its application in different contexts. Results Presenting individual case studies has highlighted how purposive sampling can be integrated into varying contexts dependent on study design. The sampling strategies clearly situate each study in terms of trustworthiness for data collection and analysis. The selected approach to purposive sampling used in each case aligns to the research methodology, aims and objectives, thus addressing each of the aspects of rigour. Conclusions Making explicit the approach used for participant sampling provides improved methodological rigour as judged by the four aspects of trustworthiness. The cases presented provide a guide for novice researchers of how rigour may be addressed in qualitative research. Natural products derived from plants as a source of drugs. Jan 2012 Ciddi Veeresham Natural products as potential drug permeation enhancer in transdermal drug delivery system Jan 2014 ARCH DERMATOL RES Umesh Patil Richa Saraogi Permeation enhancers are defined as substances that are capable of promoting penetration of drugs into skin and transdermal therapeutic systems offers a more reliable mean of administering drug through the skin. Skin is a natural barrier so it is necessary to employ enhancement strategies to improve topical bioavailability. This review explores that natural products have got potential to enhance the permeation of the drug through skin by reversibly reducing the skin barrier resistance. The use of natural products is the most reliable means of permeation enhancement of transdermally administered drugs and permits the delivery of broader classes of drugs through the stratum corneum. They are safe, non-toxic, pharmacologically inert, non-irritating, and non-allergenic to use as permeation enhancers. The present review initially highlights the current status of natural products on the basis of SAR studies which have shown significant enhancer activities. African aloe ecology: A review Stephen Ross Cousins E. T.F. Witkowski Intercropping ginger and turmeric with poplar (Populus deltoides ‘G-3’ Marsh.) May 1993 S. C. Jaswal V. K. Mishra K. S. Verma Performance of two rhizomatous crops i.e. ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) was investigated under rainfed conditions in pure stands and as intercrops with 5-year-old poplars planted at three spacings viz., 5×5 m, 5×4 m and 5×3 m. The average illumination below the canopies was 53, 46 and 38% of incident radiation, respectively. Both crops performed better as intercrops than as pure stands. Survival was inversely correlated to light intensity. Plant height, tillers per plant and leaves per plant in ginger and leaf length and leaf breadth besides plant height in turmeric were significantly enhanced when intercropped. The rhizome length, rhizome breadth, yield per plant and yield per ha in ginger exceeded under poplars but showed a drastic reduction under the closest poplar spacing. In turmeric, the trend for the first two characters was the same, whereas yield per plant as well as yeild per ha were slightly greater in the open than under 5×3 m spacing. Dry matter content varied significantly with spacing. For quality parameters, only oil content in ginger and oleoresin in turmeric showed significant differences. The cultivation of turmeric proved more remunerative than ginger. Among the poplar spacings, 5×4 m for ginger and 5×5 m for turmeric were delineated as the best spacings. Prioritization of medicinal plants on the basis of available knowledge, existing practices and use value status in Uttaranchal, India Chandra Prakash Kala Nehal Farooquee In order to understand the pattern of indigenous uses of medicinal plants available in the Uttaranchal state of the Indian Himalaya, this study was undertaken through literature survey and fieldwork in various parts of the state. A list of all the major and most of the lesser categories of ailments was prepared and categorized with the help of medical practitioners. A total of 300 plant species used in curing 114 ailments prevailing in various ethnic and non-ethnic communities of Uttaranchal were documented. These 114 ailments were further grouped into 12 broad classes of diseases in order to project the indigenous uses of medicinal plants for various ailments. It was found that herbs contributed the highest number of medicinal plants (65%), followed by shrubs (19%) and trees (16%). The maximum number of plant species were used to cure generalized body aches and colic, followed by gastrointestinal and dermatological problems. Vitex negundo was the most important species, used for the treatment of more than 48 ailments. Azadirachta indica, Woodfordia fruticosa, Centella asiatica, Aegle marmelos, Cuscuta reflexa, Butea monosperma, Phyllanthus emblica, and Euphorbia hirta were among other important medicinal plants based on their high use values. The underground parts of the plant were used in the majority of cases. Of 300 medicinal plants, 35 were rare and endangered species, of which about 80% was restricted to the high altitude alpine region of Uttaranchal Himalaya. A priority list of 17 medicinal plant species was prepared on the basis of endemism, use value, mode of harvesting and rarity status. Strategies for long-term conservation of these valuable medicinal plants are discussed. Growth and yield of turmeric (Curcuma longa L.) intercropped in poplar (Populus deltoides Bartram ex Marshall) plantation at Punjab Gill Medicinal Plants in India: An Assessment of their Demand and Supply. National Medicinal Plants Board, Ministry of AYUSH, Government of India G S Goraya D K Ved Goraya, G.S., Ved, D.K., 2017, Medicinal Plants in India: An Assessment of their Demand and Supply. National Medicinal Plants Board, Ministry of AYUSH, Government of India, New-Delhi and Indian Council of Forestry Research & Education, Dehradun, India. Cultivation of Ocimum Jat Turmeric extension pamphlet E Jayashree K Kandiannan D Prasath B Sasikumar C M Kumar V Srinivasan R Suseela Bhai C K Thankamani Jayashree, E., Kandiannan, K., Prasath, D., Sasikumar, B., Senthil Kumar, C.M., Srinivasan, V., Suseela Bhai, R., Thankamani, C.K., 2015, Turmeric extension pamphlet. ICAR-Indian Institute of Spices Research, Kozhikode, India, pp. 1-12. Executive brief, district-wise cost of cultivation of important crops in Punjab M Kaur S Singh J Singh J M Singh H S Kingra J Sachdeva Expert consultation on promotion of medicinal and aromatic plants in the asia-pacific region: proceedings Jan 2013 1-259 R Paroda S Dasgupta B Mal S P Ghosh S K Pareek Paroda, R., Dasgupta, S., Mal, B., Ghosh, S.P., Pareek, S.K., 2013, Expert consultation on promotion of medicinal and aromatic plants in the asia-pacific region: proceedings, Bangkok, Thailand, pp. 1-259. Influence of intercropping corn and Aloe vera on some characteristics of Aloe vera. Research Trend, Biological WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants Salud Organización Mundial De La Organización Mundial de la Salud, World Health Organization, 2003, WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants. World Health Organization, Geneva, Switzerland, pp.1-67. Marketing system and agricultural development in Punjab. Economic Transformation of a Developing Economy Springer Sidhu, M.S., 2016. Marketing system and agricultural development in Punjab. Economic Transformation of a Developing Economy Springer. Singapore 141-164. Good Agricultural Practices: A prequisite approach for enhancing the quality of Indian herbal medicines. Biomedical A comparative review on the standard quality parameters of turmeric, Indian Growth and yield of turmeric (Curcuma longa L.) intercropped in poplar (Populus deltoides Bartram ex Marshall) plantation at Punjab Jan 2011 40-42 B S Gill A Singh N Gandhi Jan 2014 R S Jat Nagaraja Reddy Jan 2015 R S Jat Nagaraja Reddy H R Ganjali M Dahmardeh Jan 2018 1-4 P A Singh A Baldi Jan 2006 1-32	https://www.researchgate.net/publication/369991210_Farm-level_medicinal_plants_production_and_economic_analysis_in_the_Indian_state_of_Punjab
World War II Servicemen's Correspondence Collection, 1941-1945 \| SDSUnbound Collection Description A collection of approximately 5,000 original letters sent by San Diego State students on active duty in the armed forces during World War II, or their families, to Professor Lauren Post who compiled and distributed the Aztec News Letter. Immediately after Pearl Harbor and as the War progressed, Aztec students were soon scattered all over the globe, disconnected and separated from their college, their friends, their loved ones, and their former lives. In the spring of 1942, Dr. Post, professor of geography from 1937-69 and a veteran of World War I, decided to try a "news service experiment" intended to provide information about the locations of current and former Aztecs dispersed around the world. The Aztec News Letter thus became a crucial lifeline throughout the War for Aztec servicemen and women facing difficulty and danger wherever they were stationed. Lauded as one of the first of its kind in the nation, the Aztec News Letter premiered in May of 1942 and was released monthly until its 48th issue in March of 1946. By then, The Aztec News Letter circulated to more than 3,000 service and home front readers. The project and the letters that made it possible would forever characterize the Aztec spirit during wartime. Not only are the letters historically significant; they also offer a unique perspective about San Diego State during a global crisis. Back to top Grid view List view Letter from Paul W. Casad, 1942-06 Letter from Paul W. Casad, 1942-07 Letter from Paul W. Casad, 1942-09 Letter from Paul W. Casad, 1942-10 Letter from Paul W. Casad, 1943-03 Letter from Paul W. Casad, 1943-09 Letter from Paul W. Casad, 1944 Letter from Paul W. Casad, 1945-03 Letter from Paul W. Casad, 1945-10 Bookmark Bookmarks: - Select bookmark list - My Default List Limit by Date 1942-06-05 (1) + - 1942-07-06 (1) + - 1942-09-02 (1) + - 1942-10-30 (1) + - 1943-03-14 (1) + - 1943-09-21 (1) + - 1944-09-16 (1) + - 1945-03-10 (1) + - 1945-10-01 (1) + - Type Letter (9) + - Topic Football (1) + - Fraternities (2) + - Line-crossing ceremonies (1) + - Shellbacks (1) + - World War II -- Campaigns -- Pacific (2) + - World War II -- Censorship (1) + - World War II -- Combat Air Service Unit, Advanced 42 (1) + - World War II -- Damage (1) + - World War II -- Military -- Rations (1) + - World War II -- Navy -- V-12 College Training Programs (1) + - World War II -- Training (5) + - Show more Geographic subject(s) Carlsbad Caverns National Park (New Mexico) (1) + - Dolores Airport (Philippines) (1) + - Lindbergh Field (San Diego) (1) + - South Pacific (1) + - Geographic Area Asia -- Philippines -- Eastern Samar -- Guiuan (1) + - North America -- United States -- California -- San Diego -- Liberty Station (1) + - North America -- United States -- California -- San Diego -- San Diego International Airport (1) + - North America -- United States -- Texas -- College Station -- Texas A&M University (2) + - North America -- United States -- Texas -- Corpus Christi -- Naval Air Station Corpus Christi (2) + - Pacific Ocean (1) + - Subject names Bartholdi, John J. (1) + - Brown, Bud (1) + - Brown, Forrest (1) + - Brown, Matthew (1) + - Cannon, Charles (1) + - Conn, William (1) + - Eckle, Andy (1) + - Eisert, Fred E. (2) + - Faris, Bill (1) + - Frisbie, Ralph (1) + - Gellein, Raymond L. (1) + - Ghio, Anthony A. (2) + - Godfrey, Forrest J. (1) + - Hall, Joseph Roy (1) + - Harwood, Robert (1) + - Kinsella, James J. (1) + - Lane, Humphrey P. (1) + - McQuaid, Ralph, Jr. (1) + - Morgan, William (1) + - Rice, Thomas (1) + - Rice, Tom (1) + - Richardson, Alan (1) + - Show more Organization Consolidated Vultee Aircraft Corporation (Convair) (1) + - Tau Delta Chi (1) + - United States Army Air Forces (1) + - United States Navy (8) + - United States Navy Radio Materiel School (3) + - United States Navy, Combat Air Service Unit, Advanced 42 (1) + - Language en_US (9) + -	https://digitallibrary.sdsu.edu/islandora/object/sdsu:WWIIServiceLetters?f%5B0%5D=-mods_subject_hierarchicalGeographic_area_ms%3A%22North%5C%20America%5C%20%5C-%5C-%5C%20United%5C%20States%5C%20%5C-%5C-%5C%20Alaska%5C%20%5C-%5C-%5C%20Sitka%5C%20%5C-%5C-%5C%20Sitka%5C%20Naval%5C%20Operating%5C%20Base%5C%20and%5C%20U.S.%5C%20Army%5C%20Coastal%5C%20Defenses%22&f%5B1%5D=-facetlms_mods_unified_author%3A%22Ball%2C%5C%20Richard%5C%20S.%22&f%5B2%5D=facetlms_mods_unified_author%3A%22Casad%2C%5C%20Paul%5C%20W.%22
Microorganisms \| Free Full-Text \| Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3′-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s. Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols Hanbit Song 1,2,† , Pyung-Gang Lee 1,2,3,† , Hyun Kim 1,2 , Uk-Jae Lee 1,2 , Sang-Hyuk Lee 1,2 , Joonwon Kim 1,2 and Byung-Gee Kim 1,2,4,5,* School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Korea * Author to whom correspondence should be addressed. † Hanbit Song and Pyung-Gang Lee contributed equally to this work. Microorganisms 2021 , 9 (9), 1866; https://doi.org/10.3390/microorganisms9091866 Received: 2 July 2021 / Revised: 27 August 2021 / Accepted: 27 August 2021 / Published: 2 September 2021 (This article belongs to the Special Issue Advanced Biotechnology of Microbial Enzymes ) Abstract : Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium ( Bm Ty) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of Bm Ty toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3′-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s. Keywords: plant polyphenols ; flavonoids ; monooxygenases ; tyrosinases ; ortho -hydroxylation ; biotransformation 1. Introduction Tyrosinase is a di-copper containing oxygenase that catalyzes phenolic compounds into catechols, and subsequent its catecholase activity converts the catechols into the corresponding quinones, finally leading to the synthesis of biological melanins [ 1 , 2 , 3 ]. The widespread presence of tyrosinases across bacteria, animals, plants, and fungi enables us to generate a vast library of the oxygenases characterized by broad spectra of molecular structures, substrate specificities, and catalytic properties. Above all, tyrosinase hydroxylates L-tyrosine (i.e., monophenolic substrate) at its ortho -position of its hydroxyl group and further oxidizes the formed di-hydroxy catechol compound, i.e., L-3,4-dihydroxyphenylalanine(L-DOPA), into the corresponding quinone compound. The enzyme usually has conserved di-Cu-coordinating six histidine residues as their essential structural homology sequences. Among its various applications, phenol detection/treatment in wastewater and melanin pigment synthesis are worth to noting as major ones. Recently, it was reported that some tyrosinases could become efficient biocatalysts to prepare large scale ortho -hydroxylated plant polyphenols [ 4 , 5 , 6 ]. In the biotransformation, monophenolase activity was selectively used for ortho -hydroxylation of daidzein, genistein, and resveratrol, while diphenolase (catecholase) activity was suppressed by catechol chelation by borate buffer and excess usage of antioxidants such as ascorbic acid. For diphenolase activity, melanins or melanin-like compounds are final products that can be applied for MRI reagents [ 7 ], photo-thermal/acoustic materials [ 8 , 9 ], or even structural coloring materials [ 10 ]. Moreover, the quinolic intermediate generated through diphenolase activity can form hydrogels from proteinous polymers (e.g., collagen, gelatin) and tyramine-modified carbohydrate polymers (e.g., hyaluronic acid). Plant polyphenols are biological, functional, secondary metabolites composed of multiple phenolic rings. For example, quercetin and myricetin are kaempferol-derived flavonols that share a hydroxyl group at C3 and have additional catechol or gallol moieties at their B-ring, respectively. The roles of polyphenols in the plant include defending themselves from herbivores through bitter tastes, bridging a chemical communication with symbiotic microbes as transcriptional regulators having binding affinity to its corresponding receptors, and generating flower coloration [ 11 , 12 , 13 ]. Apart from such natural roles, polyphenols also display various supplementary dietary effects such as antioxidative, antimicrobial, or anticancer activities. For example, myricetin is one of the most important polyphenols categorized as a flavonol, giving diverse biological benefits such as anticancer, antiviral, antithrombotic, anti-atherosclerotic, and antidiabetic functions. In a clinical study, relatively low incidence rates of several chronic diseases were significantly associated with high intake of myricetin as well as other flavonoids comprising quercetin, kaempferol, naringenin, and hesperetin. Additionally, a recent study found that myricetin is effective in treating the infection of the Ebola virus [ 14 ]. Due to such medicinal and clinical values of these polyphenols, preparations of polyphenolic substances such as various nutraceuticals or drugs have been attempted for a long time, using solvent extraction from natural plant resources [ 15 , 16 , 17 ]. However, solvent extraction has issues on labor-intensive gathering natural resources, usage of a large amounts of non-ecofriendly organic solvents and irregularity of bio-active ingredients depending on the harvest conditions of sources [ 18 , 19 ]. Biosynthesis of polyphenolic compounds in the industry might be an overcoming solution to these issues. Recently, quite remarkable but partial successes have been made in the production of flavonoids, isoflavonoids, stilbenoids, and their derivatives using either microbial biotransformations or microbial production systems with metabolic engineering [ 20 , 21 , 22 , 23 , 24 ]. Still, some polyphenolic derivatives can hardly be prepared for practical commercial usage. One of the most challenging compounds is myricetin which naturally comes from the biosynthetic pathway of flavonols, where a plant cytochrome P450 (CYP), CYP75A, known as flavonoid 3′,5′-hydroxylase (F3′5′H), conducts two consecutive hydroxylation at C-3′ and 5′ of kaempferol, converting it into myricetin [ 25 ]. Recently, a study reported that a Saccharomyces cerevisiae strain comprised of plant-derived flavonoid 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), F3′5′H, and flavonol synthase (FLS) succeeded to produce 145 mg/L of myricetin from naringenin after 3 days of cultivation [ 21 ]. However, these P450s are NAD(P)-dependent, so they only work in vivo or with the supplement of high concentration of NAD(P). Furthermore, their poor functional expression and low catalytic activities of the CYP enzymes in heterologous microbial hosts gave poor bioconversion yields. Except for P450s, there have been no other alternative biocatalysts synthesizing such kinds of gallol flavonoids. Herein, a remarkable success in the synthesis of catechol and subsequent gallol from kaempferol is reported using a bacterial tyrosinase ( Figure 1 ). In the efficient synthetic processes for such valuable polyphenolic products, the high instability of the plant polyphenols under the tyrosinase reaction condition was a primary obstacle. To overcome such problems, the biotransformation was attempted under the mild acidic condition of pH 6.0, considering the optimal pH of the tyrosinase reaction (ca. pH 8.0) and the auto-oxidation of myricetin or quercetin often induced under alkaline conditions. Thus, kaempferol was converted into quercetin with a yield of 60% equivalent moles of initial kaempferol, and myricetin production was also possible, up to 57 μM (ca. 6% yield and 18 mg/L/h productivity for 1 mM kaempferol) with consecutive two-step oxidative hydroxylations. In addition, the synthesis of catechol and gallol derivatives from apigenin or naringenin was confirmed with MS identification, which implied a broad substrate window of the bacterial tyrosinase. 2. Materials and Methods 2.1. Materials Kaempferol, quercetin dehydrate, and myricetin were purchased from Toronto research chemicals (Toronto, ON, Canada), Riedel-de Haën (Honeywell, Charlotte, NC, USA), and Fluka (Honeywell, Charlotte, NC, USA), respectively. The other flavonoids, apigenin, and rac -naringenin and the other compounds were purchased from Sigma Aldrich (Merck KGaA, Darmstadt, Germany). All flavonoids were dissolved in specific amounts of dimethyl sulfoxide (DMSO) with 50 or 100 mM as stock concentrations. As microbial sources for cloning of Kg Ty and Pg Ty, Kitasatospora griseola MF730-N6 and Photobacterium galatheae S2753 were kindly provided by Russell G. Kerr (University of Prince Edward Island, Charlottetown, PE, Canada) and Lone Gram (Technical University of Denmark, Kongens Lyngby, Denmark), respectively. Burkholderia cepacia KCCM 41422 (ATCC 25416) for cloning of Bc Ty was obtained from the Korean Culture Center of Microorganisms. 2.2. Measurement of Flavonoid pH Stability In the sample volume of 400 μL, 500 μM of final quercetin or myricetin concentration was dissolved in the 50 mM buffer of pH 5.0~8.0 containing 1% ( w / v ) polyvinylpyrrolidone (PVP), and the samples were incubated at 37 °C with gentle shaking (200 rpm). Citrate-phosphate buffer (pH 5.0~7.0) and Tris-HCl buffer (pH 8.0) were used for the buffering system and PVP was added to dissolve flavonoids [ 26 ]. Sampling time points were at 1, 2, 6, and 24 h after the incubation started. For quercetin, 40 μL of aliquots were five times diluted with 160 μL of HPLC grade methanol and these diluted samples were analyzed by HPLC. For myricetin, 50 μL of aliquots were diluted four times with 150 μL of DW, and extracted with 1 mL of ethyl acetate (EA). EA part of 500 or 300 μL was retrieved and vacuum evaporated. These evaporated samples were dissolved with 100 μL of HPLC grade methanol and analyzed by HPLC. 2.3. Tyrosinase Activity Assay Enzyme expression and purification were conducted with slight modifications of previous work [ 27 ]. Six bacterial tyrosinases had been transformed in E. coli BL21 (DE3) and their cell stocks were used to express each tyrosinase ( Table S1 ). Each cell was inoculated in 1.1 mL of Luria-Bertani lysogeny broth with the desired antibiotic (LB) depend on each vector and cultured under 37 °C with gentle shaking (200 rpm) for 8 h. Each 0.2 mL cultured inoculum was added to 10 mL of LB and incubated under 37 °C with gentle shaking until the turbidity (OD 600 ) became 0.8~1.1. At the end of incubation, isopropyl β-D-1-thiogalactopyranoside (IPTG) induction was conducted in the incubator of 18 °C, 200 rpm for overnight (ca. 18 h) with the addition of 0.5 mM CuSO 4 and 0.2 mM IPTG as final concentrations. After IPTG induction, each cell was collected and washed with phosphate-buffered saline (PBS) using a refrigerated centrifuge operated at 4000 rpm and 4 °C. The washed cells were resuspended in 1 mL of 50 mM Tris-HCl buffer pH 8.0 with 0.25 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspensions were sonicated and centrifuged at 16,000 rpm, 4 °C. The supernatants were injected in columns packed with Ni-NTA agarose bead (Qiagen Korea Ltd., Seoul, Korea) and the 6x His-tagged tyrosinases were purified. The purified tyrosinases were dialyzed by ultrafiltration with Amicon Ultra-15 (Merck Millipore, Ltd., Seoul, Korea). To evaluate the activities of tyrosinases against kaempferol or quercetin at acidic pH range of 4.0~6.0, a colorimetric assay was exploited with the aid of MBTH. In each well of 96-well plate, 200 μL of 50 mM buffer (pH 4.0: citrate buffer, pH 5.0, 6.0: citrate-phosphate buffer) solution mixture containing 10 μM CuSO 4 , 5 mM MBTH, 0.5 μM tyrosinase, 1% ( w / v ) PVP and 0.5 mM kaempferol or quercetin was examined. For the blank control, 50 mM, pH 8.0 Tris-HCl buffer was added instead of tyrosinase. These 200 μL mixtures were prepared by mixing two parts: 50 μL of 4× substrate mixture containing 4% ( w / v ) PVP 40k and 2 mM kaempferol or quercetin and 150 μL of tyrosinase mixture, which contained the others to complete the mixture. These two parts were incubated under 37 °C separately for 10 min and mixed. Soon after, absorbance at 505 nm of each well was measured for an hour, with an interval of 30 s, using a UV/Vis spectrophotometer (SPECTROstar Nano; BMG Labtech GmbH, Ortenberg, Germany). In addition, by measuring the absorbance at 475 nm, which is the maximal absorption wavelength of dopachrome, activities on L-tyrosine and L-DOPA of Bm Ty were obtained under pH range of 5.0~8.0 [ 28 ]. For the dopachrome assay, in each well of 96-well plates, 200 μL of 50 mM buffer (pH 5.0~7.0: citrate-phosphate buffer, pH 8.0: Tris-HCl buffer) solution mixture containing 10 μM CuSO 4 , 0.1 μM Bm Ty, and 1 mM L-tyrosine was used. Similar to the above procedure, a blank control was prepared. The absorbance at 475 nm was measured for 30 min with an interval of 30 s. The amounts of Bm Ty needed for conversion of 1 μmol L-tyrosine (monophenolase activity) or L-DOPA (diphenolase activity) per minute were defined as 1 enzyme unit (U) at different pHs, and the enzyme units of monophenolase activity deduced from this activity test ( Table 1 ) utilized for further experiments. 2.4. Kaempferol Conversion Using BmTy at Various pHs In 1 mL reaction volume, 0.2 mM kaempferol, 1% ( w / v ) PVP 40k, 1% ( v / v ) DMSO, 3 mM L-ascorbic acid, 2 μM CuSO 4 and 50 mU/mL Bm Ty (427, 216, 133 and 181 nM enzyme at pH 5, 6, 7 and 8, respectively, Table 1 ) were added into 50 mM buffer (pH 5.0~7.0: citrate-phosphate buffer, pH 8.0: Tris-HCl buffer) as final concentrations. Aliquots of 100 μL were vortexed with 400 μL EA at specific sampling points in time, and 200 μL of EA parts were retrieved and dried. The residues were dissolved with 100 μL MeOH and analyzed with HPLC. 2.5. MS Identification of Various Ortho-Hydroxylated Products Three polyphenol substrates, kaempferol, apigenin, rac -naringenin, were used to assess the hydroxylation ability of Bm Ty on broad substrates. For kaempferol, 500 μL of reaction mixture containing 0.2 mM kaempferol, 3% ( w / v ) PVP 40k, 25 mM L-ascorbic acid (LAA), 10 μM CuSO 4 , 1 μM Bm Ty and 50 mM citrate-phosphate buffer pH 6.0 was incubated at 37 °C with shaking (200 rpm) for 2 h. For apigenin, all the other reaction condition were the same as kaempferol sample, but 0.2 mM apigenin, instead of kaempferol, and 5% ( w / v ) PVP 40k were used. For rac -naringenin, 400 μL of reaction mixture containing 1 mM rac -naringenin, 5% ( w / v ) PVP 40k, 25 mM L-ascorbic acid, 10 μM CuSO 4 , 1 μM Bm Ty and 50 mM citrate-phosphate buffer pH 6.0 was incubated at 37 °C with shaking (200 rpm) for 3 h. In the case of kaempferol and apigenin samples, 400 μL of aliquot was retrieved and diluted with 1200 μL of DW. Each diluted sample was extracted with 2 mL of EA and 1.6 mL of EA was retrieved and vacuum-evaporated. For the rac -naringenin reaction mixture, 50 μL of aliquot was retrieved and diluted with 150 μL of DW. This diluted sample was extracted with 1 mL of EA and 0.8 mL of EA was retrieved and vacuum-evaporated. The evaporated samples from kaempferol or rac -naringenin reactions were dissolved in 100 μL of EA and added 2 μL of N , O -Bis(trimethylsilyl)trifluoroacetamide (BSTFA) to derivatize trimethylsilyl (TMS) group. The TMS derivatization was conducted under 37 °C for 20 min. The 0.5 or 1 μM of authentic references were also dissolved in EA and these authentic samples were TMS-derivatized, as with other analyzed samples. These derivatized samples were analyzed with GC-MS. Evaporated residue from apigenin reaction was dissolved in 200 μL of methanol and filtered with 3 kDa cut-off centrifugal filter (Merck). The flow-through was analyzed with HPLC-ESI-MS/MS. For GC-MS analysis, TRACE GC Ultra gas chromatograph (GC) coupled with ITQ1100 ion trap mass detector (Thermo) was used. In GC, a nonpolar capillary column (5% phenyl methyl siloxane capillary 30 m × 250 μm i.d., 0.25-μm film thickness, TR-5 ms) was equipped. Each 1 μL sample was injected with the splitless mode of 0.8 min splitless time. The oven schedule started with the initial temperature of 65 °C and held for one minute, and then the oven temperature increased to 250 °C, with the ramp rate of 30 °C/min. After 3 min of hold time at 250 °C, the second ramp proceeded with the rate of 5 °C/min until the temperature reached 300 °C. Finally, the temperature was held for 20 min at 300 °C. The ion source temperature, flow rate of carrier gas (Helium), and electron energy of EI were 230 °C, 1 mL/min, and 70 eV, respectively. For HPLC-ESI-MS/MS analysis, TSQ Quantum Access Max coupled with Accela HPLC system (Thermo) equipped with the COMOSIL 5C 18 -AR-II column (5 μm particle size, 4.6 mm I.D. × 150 mm; Nacalai Tesque, Kyoto, Japan) was used. In HPLC, eluent of 65% water with 0.1% formic acid and 35% acetonitrile with 0.1% formic acid passed through the C 18 column with the flow rate of 0.4 mL/min. Each 10 μL sample was injected into HPLC and analyzed with ESI-MS/MS. The condition for mass spectrometry was as follows: spray voltage, 3.0 kV; vaporizer temperature, 573 K; capillary temperature, 593 K; sheath gas pressure, 30 psi; aux gas pressure, 10 psi. ESI-MS/MS was operated in positive ion mode and two scan events occurred per acquired ions. The first scan event was a full scan in the mass range of m / z : 200~600. The second scan was a dependent scan that the most intense ion in the first scan was fragmented with the collision energy of 35 eV and scanned. 2.6. Biosynthesis of Quercetin and Myricetin Using BmTy As a reaction mixture solution, 400 μL of 50 mM citrate-phosphate buffer (pH 6.0) solution containing 1 μM Bm Ty, 10 μM CuSO 4 , 3% ( w / v ) PVP 40 k, 25 mM LAA, and 0.2~2 mM kaempferol were used. DMSO was adjusted to make 4% ( v / v ) of a final DMSO concentration for all the samples. One-hour reaction was performed in an orbital shaker with 200 rpm at 37 °C. The reaction was finished by EA extraction. For the sample extraction, 50 μL aliquot of the reaction mixture was mixed with 150 μL DW and then extracted with 1 mL EA. EA part of 800 μL was retrieved and vacuum evaporated. This dried sample was dissolved with 100 μL of HPLC-grade methanol and analyzed with HPLC. 2.7. HPLC Analysis For HPLC analysis, HPLC (YL9100 HPLC; YoungLin, Anyang, Korea) equipped with the COMOSIL 5C 18 -AR-II column was used. Each 10 μL sample was injected and the flow rate was 0.7 mL/min. The elution ratio of water with 0.1% ( v / v ) trifluoroacetic acid to acetonitrile was 65:35 (volume ratio). The flow-through from the C 18 column was measured by UV detector at the wavelength of 376 nm. The calibration curves of flavonoids were obtained by measuring 10, 100, 500, and 1000 μM samples dissolved in HPLC grade methanol. By comparing these calibration curves, the concentration of each sample was measured for quantification analysis. 2.8. Preliminary Enzyme Deactivation Tests with the Incubation of Flavonols In each well of 96-well plates, 280 μL of 50 mM citrate-phosphate buffer (pH 6.0) solution containing final concentrations of 2 μM CuSO 4 , 0.5 mM kaempferol, quercetin or myricetin, 0.5% ( v / v ) DMSO, 4% ( w / v ) PVP 40 k, and 0.5 μM Bm Ty was added. Flavonols were omitted for negative control and 0.5 mM L-tyrosine was added instead of flavonols for positive control. For blank, flavonols, Bm Ty, and L-tyrosine were all omitted (Figure 8A). For Figure 8B, almost the same components were added except final concentrations of 0.1~0.4 mM kaempferol, quercetin or myricetin, 0.8% ( v / v ) DMSO and 0.4 μM Bm Ty were used, instead. They were all incubated at 37 °C, and at specific incubation points in time, the incubated solutions were diluted five times (for Figure 8A) or ten times (for Figure 8B) in the buffers containing 50 mM, pH 8.0 Tris-HCl buffer, and 1 mM L-tyrosine as final concentrations and the tyrosinase reaction was started. The reactions were proceeded at 37 °C and monitored at the 475 nm wavelength for dopachrome assay, and initial rates were calculated. The initial rates were treated as residual initial rates ( V res ) at specific incubation times ( t i ) and regressed using the following equation to obtain the ] deactivation coefficients ( k d ), with an assumption such that the concentration of L-tyrosine is far above K M, such that the initial rate of the native enzyme is identical to V max . ln V res V 0 = k d t i where V 0 is the residual initial rate at t i = 0. Based on the following chemical equations and assuming that the concentration of an incubated deactivator [D] is constant during incubation (because of its high concentration compared to [E]), E → k d 1 E d , E + D → k d 2 E d k d can be more precisely expressed with the equation shown below. k d = k d 1 + k d 2 D k d1 is spontaneous deactivation constant of the enzyme and k d2 is suicide deactivation constant of the enzyme by a deactivator. Therefore, if a suspected deactivator deactivates the enzyme in a suicidal manner, the k d value will be changed linearly according to [D]. 2.9. BmTy Inhibition Kinetics of Quercetin In 96-well plate, each well of 200 μL aqueous solution was added to make a final solution of 50 mM of Tris-HCl buffer pH 8.0, 10 μM of CuSO4, 0~100 μM of quercetin, 0.1% ( v / v ) of DMSO, 25~100 μM of L-tyrosine and 0.1 μM of Bm Ty. Michaelis–Menten kinetics were measured by monitoring 475 nm dopachrome wavelength and recording the initial rate by varying quercetin and L-tyrosine concentrations. Each sample was triplicated, and the data were shown as averages with standard errors. The apparent k cat ( k cat app ) and K M ( K M app ) values were obtained by non-linear regression using the following equation. V i = k cat app E t S i K M app + S i V i , is the initial reaction rate, and [E] t and [S] i are initial Bm Ty concentration and initial L-tyrosine concentration, respectively. Inhibition constants were obtained from two apparent constants by regressing the following equations. K M app K M k cat k cat app = 1 + I K I 1 , k cat k cat app = 1 + I K I 2 [I], K I1 and K I2 are quercetin concentration, competitive inhibition constant, and uncompetitive inhibition constant, respectively. 3. Results 3.1. Measurement of pH Stability of Quercetin and Myricetin In flavonoids oxidation reaction such as biosynthesis of ortho -hydroxylated plant polyphenols (i.e., quercetin and myricetin) using tyrosinase, it was well known to a certain extent that auto-oxidation of ortho -hydroxylated polyphenols at alkaline pH conditions would be the main problem [ 29 , 30 , 31 , 32 , 33 ]. Therefore, we first measured the stability of the quercetin and myricetin as representatives of catechol and gallol compounds, respectively, within the pH range of 5 to 8. Although kaempferol was stable in the neutral aqueous solution of up to pH 7.0 for 4 days [ 34 ], quercetin and myricetin were susceptible to oxidation particularly under alkaline-to-neutral conditions ( Figure 2 ). Quercetin was stable in the moderately acidic condition (ca. pH 5 to 6), while it was rapidly oxidized at pH 7 to 8 (i.e., 40% of quercetin was oxidized after 24 h at pH 8). Moreover, myricetin was more vulnerable at neutral or basic pH than quercetin. At above pH 7, most of the myricetin was oxidized within 24 h ( Figure 2 ). The results imply that autooxidation of quercetin and myricetin at neutral and basic pH is attributed to alkali-sensitive catechol and gallol moieties, respectively, which are unique functional groups of those flavonols [ 35 , 36 ]. As a result, the acidic condition is desirable for efficient synthesis of the catechol and gallol, also suggesting that potentially good flavonol oxygenases should exhibit significant catalytic activity under the acidic pH (i.e., ≤6). 3.2. Screening Bacterial Tyrosinases for Polyphenol Conversion under Acidic Condition Our group has searched various tyrosinases from diversified resources for specialized applications. One of them is a tyrosinase from Streptomyces avermitilis (MelC2, and here so-called, Sa Ty). This tyrosinase was reported to have substrate specificity not only on some small molecules like L-tyrosine or trans -resveratrol [ 5 , 37 ] but also on bio-macromolecules like gelatin or tyramine-modified polysaccharides, such as chitosan and hyaluronic acid [ 38 ]. Other tyrosinases from Kitasatospora griseola ( Kg Ty) and Photobacterium galatheae ( Pg Ty) were selected because they are the two representatives of a few subgroups in the cladogram of bacterial tyrosinases built with the aid of Subgrouping Automata program [ 39 ]. Most tyrosinases have their optimal activity in neutral-to-slightly alkaline pH (ca. 7~8). Recently, a tyrosinase from Burkholderia thaliandensis ( Bt Ty), which showed potent activity at low pH (ca. pH 4–5) with tetrameric structure, was found [ 40 ]. Since Bt Ty successfully converted daidzin (isoflavone glycoside), resveratrol (stilbene), and phloretin (chalcone) into their corresponding hydroxylated products, respectively, it was considered as a potential catalyst for the hydroxylation of kaempferol to produce quercetin as well as myricetin. In an effort to discover other acidic tyrosinases, a tyrosinase from Burkholderia cepacia ( Bc Ty), which had high sequence homology with Bt Ty, was found. Along with the Bm Ty which had already been proven to show good activities on various polyphenols [ 40 ], the above-mentioned five tyrosinases were assessed for flavonol oxidation screening. However, all the tyrosinases except Bm Ty showed marginal activities for kaempferol and quercetin according to MBTH assay (quinone-detecting method) at pH 4 to 6 ( Figure 3 ). In the case of Bm Ty, because its optimal pH was around pH 8 [ 40 ] the flavonol-hydroxylating activity was relatively higher at slightly acidic pH (i.e., 6) than at acidic pH (i.e., 4 or 5). Since all the rest of the tyrosinases showed marginal activity, Bm Ty was chosen to perform further examination. Next, monophenolase and diphenolase activities of Bm Ty were compared in various pHs to examine an optimal condition, where flavonoid hydroxylation ( k 1 ) is favorable, while quinone formation ( k 2 ) is suppressed. For that purpose, the dopachrome assay was conducted using two native substrates of tyrosinases, L-tyrosine and L-DOPA. In general, lowering the reaction pH caused more attenuation of the diphenolase activity ( k 2 ) than the monophenolase activity ( k 1 ), resulting in a higher ratio of monophenolase to diphenolase activity at acidic pH ( Figure 4 , Table 1 ). This further suggests that the use of acidic reaction conditions is favorable for flavonol synthesis because diphenolase activity was significantly suppressed, facilitating the accumulation of hydroxylated products. 3.3. Effect of pH on the Kaempferol Conversion by BmTy For the syntheses of hydroxylated compounds, two consecutive monophenolase cycles of tyrosinase were exploited ( Figure 1 ). In the first step where oxy -tyrosinase ( oxy -Ty) took part in the reaction of monophenolase cycle, ortho -hydroxylation of the monophenol compound took place and a corresponding catechol compound was generated. To complete a tyrosinase reaction cycle, met -tyrosinase ( met -Ty) subsequently oxidized the just converted catechol compound into the corresponding o -quinone compound. O -quinones are generated via two routes: one from monophenolase cycle and the other from the two rounds of oxidation steps in the diphenolase cycle. However, the o -quinones were reduced to the catechol derivatives by the reducing agent such as L-ascorbic acid (LAA). To be precise, LAA reduces o -quinones to catechols by oxidizing itself, becoming L-dehydroascorbic acid, thereby accumulating catechols intact [ 41 ]. With this reaction scheme, the effect of reaction pH on the reaction rate for the selected tyrosinase was compared using 0.2 mM kaempferol at pH 5~8 with a fixed enzyme unit of 50 mU/mL and 60 min reaction time ( Figure 5 A). The accumulation of myricetin was generally increased with the decrease in pH. Given this result, we have compared time course reactions at pH 5, where the most produced myricetin was accumulated, and pH 8, where Bm Ty showed its optimal activity ( Figure 5 B). In the reaction time courses using 0.2 mM kaempferol as an initial substrate, the accumulated amount of myricetin at pH 5 exceeded the one at pH 8 in 30 min. Especially, 51% more myricetin was accumulated at pH 5 than that at pH 8 after 60 min of reaction time. Moreover, the concentration of quercetin appeared to be much stabler compound than myricetin at alkaline pH, was higher at pH 5 than at pH 8 after 20 min of reaction time. This result indicates that pH affected the accumulated amount of hydroxylated flavonols within tyrosinase reaction time scale that acidic pH could contribute to the accumulation of the hydroxylated flavonols by preventing auto-oxidation in the buffer. In addition, the higher value of k 1 / k 2 ratios at acidic conditions would also be favorable to accumulate the products by avoiding undesirable overoxidation in quinones. 3.4. MS Identification of Tyrosinase Products Based on the above findings, we carried out ortho -hydroxylation of several plant polyphenols namely, kaempferol (flavonol), apigenin (flavone), and rac -naringenin (flavanone), using Bm Ty below pH 6. According to GC-MS or LC-ESI-MS/MS analysis, Bm Ty converted them into their corresponding catechol and gallol derivatives at B-ring ( Figure 6 , Table S2 ). For example, kaempferol was efficiently hydroxylated into quercetin (quercetin-5TMS: m / z = 662, 648, 576, 560), which was subsequently oxidized to myricetin (myricetin-6TMS: m / z = 750, 736, 664, 648). The EI-mass spectra of these flavonols agreed well with those of authentic chemicals. The overlapping fragmented ions of m / z = 394 and 281 supported the addition of hydroxyl groups in the B-ring of quercetin and myricetin, respectively. The B-ring-specific hydroxylation was also confirmed in the mass spectra of the reaction products of apigenin and naringenin, indicating luteolin (or tricetin) and eriodictyol (or 3′,5′-dihydroxynaringenin) ( Table S2 ). 3.5. Production of Quercetin and Myricetin from Kaempferol Using BmTy To determine if Bm Ty can carry out such an efficient B-ring specific hydroxylation, the effect of the initial concentration of kaempferol (0.2–2 mM) on substrate conversion and product concentration were compared ( Figure 7 ). Within the examined concentrations of kaempferol, most of the substrate was converted into quercetin and myricetin in one hour. The titer of quercetin proportionally increased with the initial kaempferol concentration, maintaining a similar yield of ca. 60%. In one-hour reaction, 57 μM of myricetin was produced from 1 mM kaempferol corresponding to 18 mg/L/h productivity. However, myricetin yield did not increase over 57 μM despite using high concentrations of kaempferol loading (ca. 2 mM). Since such flavonols were already known as strong inhibitors against tyrosinase [ 42 , 43 , 44 , 45 ], and catecholic or gallol compounds were also known to become suicide deactivators of tyrosinase [ 46 , 47 , 48 ], the increasing concentrations of the products appeared to inhibit or deactivate Bm Ty. 3.6. Inhibitory Action of Quercetin in Monophenolase Activity of BmTy To figure out the causes of such inhibition or deactivation in myricetin production, a preliminary test identifying deactivation kinetics of Bm Ty was performed ( Figure 8 ). Briefly, flavonols of interest were incubated with Bm Ty in the similar condition of ortho -hydroxylation reactions and the residual monophenolase activity was analyzed by the dopachrome assay using L-tyrosine at different incubation points in time. The monophenolase activities decreased with an exponential decay profile with time, suggesting that the deactivation of Bm Ty appeared to follow 1 st order kinetics with deactivation coefficients ( k d ). However, the deactivation coefficients did not exhibit a positive linear correlation with regard to the concentration of incubated flavonols ( Figure 8 B). This represents that suicide deactivation of Bm Ty by flavonols did not happen. Bm Ty incubated with quercetin initially showed impaired activity compared with other cases ( Figure 8 A). As a result, to examine such possibility of inhibition by quercetin, Michaelis–Menten kinetics were evaluated by varying quercetin concentration ( Figure 9 A). Within the quercetin concentration range of 0~100 μM, somewhat mixed inhibition pattern was observed in monophenolase activity of Bm Ty with a highly competitive manner of K I1 = 33.9 μM and K I2 = 417 μM ( Figure 9 B,C). The values are similar to those of the Tyrosinase from Agaricus bisphorous [ 45 ]. Therefore, it appears that the accumulated quercetin from the monophenolase reaction of Bm Ty inhibited the ortho -hydroxylation activity of Bm Ty (i.e., monophenolase activity) prohibiting myricetin production. 4. Discussion To date, tyrosinases were mainly used for medical applications, phenolic waste treatment and production of fine chemicals [ 4 , 5 , 38 , 49 , 50 , 51 ]. Our group has demonstrated that by incorporating borate chelation with a catechol intermediate generated by monophenolase activity of tyrosinase and using suitable reducing agents, several catecholic products could be successfully prepared with high yields. Outstanding examples are plant polyphenolic compounds including piceatannol and ortho -dihydroxy-isoflavones. This study demonstrated a rather simple approach of using bacterial tyrosinase for the synthesis of gallol products with commercial scale for the first time, since the enzyme is a coenzyme-free catalyst with high reaction rates unlike P450s. The gallol-producing bacterial tyrosinase, Bm Ty, was first isolated and characterized by the Fishman group [ 28 ]. According to their previous publications, Bm Ty showed broad substrate specificity for various monohydroxyphenols, dihydroxyphenols, and trihydroxyphenols, which was described with the crystal structures of the enzyme and a couple of mutants, and the broad substrate specificity has also been utilized for the preparation of functional catecholic compounds [ 4 , 52 ]. However, the previous reports on Bm Ty did not attempt to execute the production of gallols from monophenols or diphenols. Since catechols are susceptible to oxidation, it seems that acidic condition and additional reducing agents (i.e., L-ascorbate in this study) can prevent their autoxidation and over-oxidation allowing the enzyme to have a second chance of hydroxylation to intermediate diphenols, finally giving the accumulation of gallols. Taking advantage of the newly developed biocatalysis, we could demonstrate to synthesize pharmaceutically valuable flavonoids, quercetin and myricetin using one tyrosinase, Bm Ty. Using kaempferol, product quercetin was obtained with 60% yield in one-hour reaction, and the titer was up to 1.2 mM from 2 mM of kaempferol. Notably, the achieved myricetin productivity was almost 10 times higher (i.e., 18 mg/L/h) than that of the previously reported one using a plant cytochrome P450s [ 21 ]. This synthesis platform has an excellent potential for various applications, since the facile in vitro syntheses shown in this paper were possible with high efficiency without NAD(P) coenzyme. However, the final myricetin concentration in the reaction solution was restricted ( Figure 7 ). Such a drawback was caused by the product inhibition by the ortho -hydroxylation product quercetin. As quercetin was a competitive inhibitor of the mushroom tyrosinase [ 42 , 43 , 44 , 45 ], the contribution of competitive inhibition mode was higher than the uncompetitive mode when quercetin exerts mixed inhibition on Bm Ty. Escaping from competitive inhibition can be accomplished by increasing the dissociation constant of the inhibitor with mutational approaches in general cases. However, unfortunately, in this case, the inhibitor is another substrate for consecutive hydroxylation. One possible solution is increasing the turnover number ( k cat , TON) of monophenolase activity. In this way, the ortho -hydrating reaction of Bm Ty will be rescued by fast conversion of quercetin without damaging catalytic efficiency( k cat / K M ). However, it must be considered that the TON of diphenolase activity should remain constant or lower to prevent the unwanted over-oxidation of valuable products. The monophenolase to diphenolase activity ratio is another big issue in tyrosinase reactions. In general, high monophenolase activity compared to diphenolase activity is favored for achieving a high yield of hydroxylated product, because diphenolase activity causes consumption of the desired ortho -hydroxylated product and generation of quinone product, leading to the formation of melanin analogues. Actually, many reports highlighted the importance of high monophenolase to diphenolase activity ratio for various tyrosinase mutants and natural isolates [ 53 , 54 , 55 , 56 , 57 ]. As results shown, monophenolase (L-tyrosine to L-dopachrome)-to-diphenolase activity (L-DOPA to L-dopachorme) ratio of Bm Ty increased with a decrease in reaction pH, ( Figure 4 ) which is also a favored aspect for optimized mildly acidic reaction condition considering high stability of quercetin and myricetin in acidic conditions ( Figure 2 ). Such an acidic environment might help to achieve the high productivity of myricetin, despite susceptibility to autoxidation of the gallol products. The aforementioned problems may be further solved through enzyme engineering, applying selected mutations that modulate k cat and K M of individual monophenolase and diphenolase in Bm Ty homologues. A recent study reported that the monophenolase activity or diphenolase activity of type-III copper enzymes was controlled by the residues neighboring Cu-coordinating histidines such as HisB 1 + 1 and HisB 2 + 1 through modulating the basicity of histidine. Even monophenolase activity was generated by mutating aurone synthase, which did not have tyrosinase activity, and their mutational work showed plenty of mutants that vary k cat and K M of monophenolase or diphenolase activity, respectively. Throughout considering flavonol stability and optimum enzyme-reaction pH, we have constructed an easy platform of catechol and gallol biosynthesis using tyrosinase without any coenzymes in mildly acidic conditions. Despite quercetin inhibition, this platform achieved 18 mg/L/h of myricetin productivity, currently the highest value ever published. 5. Conclusions In this study, we found that pH stability was the crucial factor for the preparation of some readily oxidized flavonoids in the tyrosinase reaction. In the mild acidic condition, myricetin was facilely synthesized from kaempferol with the productivity of 18 mg/L/h in the tyrosinase one-pot reaction without any cofactors. Furthermore, some novel gallol flavonoids such as tricetin were successfully synthesized. We insist that this simple pH adjustment along with proper monophenolase activity of tyrosinase would be an efficient way to prepare versatile flavonoids hardly prepared by other ways. Supplementary Materials The following are available online at https://www.mdpi.com/article/10.3390/microorganisms9091866/s1 , Table S1: Cloning information of the six bacterial tyrosinases and used primers., Table S2: GC-MS or LC-ESI-MS/MS anaylsis of hydroxylated flavanone and flavone products. Author Contributions Conceptualization, H.S., P.-G.L. and J.K.; Data curation, H.S. and P.-G.L.; Formal analysis, H.S. and P.-G.L.; Funding acquisition, B.-G.K.; Investigation, H.S., P.-G.L., H.K., U.-J.L. and S.-H.L.; Methodology, H.S. and P.-G.L.; Project administration, H.S. and P.-G.L.; Resources, B.-G.K.; Supervision, B.-G.K.; Visualization, H.S. and P.-G.L.; Writing—original draft, H.S., P.-G.L. and B.-G.K.; Writing—review & editing, H.S., P.-G.L., H.K., U.-J.L., S.-H.L., J.K. and B.-G.K. All authors have read and agreed to the published version of the manuscript. Funding This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2017R1E1A1A01073523), Industrial Strategic technology development program, 20002734 funded by the Ministry of Trade, industry & Energy (MI, Korea) and the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: KMDF_PR_20200901_0151). Conflicts of Interest The authors declare no conflict of interest. Abbreviations MRI magnetic resonance imaging CYP cytochrome P450 NAD(P) nicotinamide adenine dinucleotide (phosphate) MS mass spectrometry HPLC high performance liquid chromatography Ni-NTA nickel-nitrilotriacetic acid UV/Vis ultraviolet/visible light GC gas chromatography ESI electrospray ionization EI electron ionization EA ethyl acetate KAE kaempferol QUE quercetin MYR myricetin References McLarin, M.-A.; Leung, I.K.H. Substrate specificity of polyphenol oxidase. Crit. Rev. Biochem. Mol. Biol. 2020 , 55 , 274–308. [ Google Scholar ] [ CrossRef ] Solomon, E.I.; Sundaram, U.M.; Machonkin, T.E. Multicopper Oxidases and Oxygenases. Chem. Rev. 1996 , 96 , 2563–2606. [ Google Scholar ] [ CrossRef ] [ PubMed ] Muñoz-Muñoz, J.L.; Berna, J.; García-Molina, M.d.M.; Garcia-Molina, F.; Garcia-Ruiz, P.A.; Varon, R.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F. Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity. Biochem. Biophys. Res. 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The two main cycles of tyrosinase, monophenolase cycle and diphenolase cycle, were represented with blue and red arrows, respectively. Some chemical species are described with abbreviations: those are: A: L-dehydroascorbic acid, AH 2 : L-ascorbic acid, Ty: tyrosinase, CAT: catechol, GAL: gallol, QUI: o -quinone. Figure 1. Schematic representation of plant polyphenol oxidation in the general tyrosinase mechanism. The two main cycles of tyrosinase, monophenolase cycle and diphenolase cycle, were represented with blue and red arrows, respectively. Some chemical species are described with abbreviations: those are: A: L-dehydroascorbic acid, AH 2 : L-ascorbic acid, Ty: tyrosinase, CAT: catechol, GAL: gallol, QUI: o -quinone. Figure 2. pH stability of quercetin ( A ) and myricetin ( B ). 500 μM of the polyphenols were incubated under respective pH at 37 °C. Each data point was indicated as mean values with standard deviations (error bars) from three independent replicates. Figure 2. pH stability of quercetin ( A ) and myricetin ( B ). 500 μM of the polyphenols were incubated under respective pH at 37 °C. Each data point was indicated as mean values with standard deviations (error bars) from three independent replicates. Figure 3. MBTH activity assay for six microbial tyrosinases against kaempferol and quercetin. Each purified tyrosinase (0.5 μM) was measured for its oxidation (or quinone formation) activity against 500 μM of substrates under mild acidic conditions. Figure 3. MBTH activity assay for six microbial tyrosinases against kaempferol and quercetin. Each purified tyrosinase (0.5 μM) was measured for its oxidation (or quinone formation) activity against 500 μM of substrates under mild acidic conditions. Figure 4. Monophenolase versus diphenolase activity assay of Bm Ty in the various pH. 1000 μM of L-tyrosine (black bar) and L-DOPA (gray bar) were used as initial substrates to measure monophenolase (tyrosine hydroxylation activity) and diphenolase activity (quinone formation activity) of Bm Ty, respectively. Figure 4. Monophenolase versus diphenolase activity assay of Bm Ty in the various pH. 1000 μM of L-tyrosine (black bar) and L-DOPA (gray bar) were used as initial substrates to measure monophenolase (tyrosine hydroxylation activity) and diphenolase activity (quinone formation activity) of Bm Ty, respectively. Figure 5. Kaempferol conversion of Bm Ty at different pHs. Concentrations of flavonols in one-hour Bm Ty reaction at pH 5~8 were shown as gray (kaempferol), blue (quercetin), and purple (myricetin) bars ( A ) and the concentrations of time course reactions at pH 5 and 8 were expressed with the same color for each compound ( B ). Concentrations at pH 8 were expressed with dashed lines and open circles and those at pH 5 were expressed with solid lines and closed circles. Figure 5. Kaempferol conversion of Bm Ty at different pHs. Concentrations of flavonols in one-hour Bm Ty reaction at pH 5~8 were shown as gray (kaempferol), blue (quercetin), and purple (myricetin) bars ( A ) and the concentrations of time course reactions at pH 5 and 8 were expressed with the same color for each compound ( B ). Concentrations at pH 8 were expressed with dashed lines and open circles and those at pH 5 were expressed with solid lines and closed circles. Figure 6. GC/MS analysis for identification of tyrosinase reaction products converted from kaempferol using Bm Ty. Two major products at RT = 18.0 min (product 1) and RT= 18.5 min (product 2) were detected, then they were identified as quercetin ( m / z = 662, product 1) and myricetin ( m / z = 750, product 2). ( A ) Gas chromatogram of the reaction sample. ( B ) MS spectrum of the product 2, myricetin. ( C ) MS spectrum of the product 1, quercetin. ( D ) MS spectrum of the standard compound of kaempferol. Figure 6. GC/MS analysis for identification of tyrosinase reaction products converted from kaempferol using Bm Ty. Two major products at RT = 18.0 min (product 1) and RT= 18.5 min (product 2) were detected, then they were identified as quercetin ( m / z = 662, product 1) and myricetin ( m / z = 750, product 2). ( A ) Gas chromatogram of the reaction sample. ( B ) MS spectrum of the product 2, myricetin. ( C ) MS spectrum of the product 1, quercetin. ( D ) MS spectrum of the standard compound of kaempferol. Figure 7. Conversion of kaempferol into quercetin (catechol) and myricetin (gallol) using Bm Ty. Purified Bm Ty (1 μM) was added to 200~2000 μM of kaempferol and conversion reactions were conducted at pH 6 and 37 °C for 1 h. Concentrations of quercetin (blue bar) and myricetin (purple bar) were measured and substrate conversions were obtained from kaempferol concentrations (line with diamond labels). Figure 7. Conversion of kaempferol into quercetin (catechol) and myricetin (gallol) using Bm Ty. Purified Bm Ty (1 μM) was added to 200~2000 μM of kaempferol and conversion reactions were conducted at pH 6 and 37 °C for 1 h. Concentrations of quercetin (blue bar) and myricetin (purple bar) were measured and substrate conversions were obtained from kaempferol concentrations (line with diamond labels). Figure 8. Deactivation kinetics of Bm Ty with 0.5 mM of kaempferol, quercetin, and myricetin. ( A ) and the deactivation coefficients with respect to 0.1~0.4 mM of putative deactivators ( B ). Each sample was represented with abbreviations, those are: neg: negative control (no putative deactivator), MYR: myricetin, QUE: quercetin, KAE: kaempferol, tyr: L-tyrosine. Figure 8. Deactivation kinetics of Bm Ty with 0.5 mM of kaempferol, quercetin, and myricetin. ( A ) and the deactivation coefficients with respect to 0.1~0.4 mM of putative deactivators ( B ). Each sample was represented with abbreviations, those are: neg: negative control (no putative deactivator), MYR: myricetin, QUE: quercetin, KAE: kaempferol, tyr: L-tyrosine. Figure 9. Mixed inhibition of Bm Ty by quercetin. Initial rates of Bm Ty were decreased when quercetin concentration was increased in the range of 0~100 μM quercetin and 37.5~300 μM L-tyrosine ( A ). Regression of two parameters derived two inversed inhibition constants, K I1 = 33.9 μM and K I2 = 417 μM, which means that quercetin is a highly competitive mixed inhibitor ( B , C ). Data were shown as averages and standard errors from triplicated samples. Figure 9. Mixed inhibition of Bm Ty by quercetin. Initial rates of Bm Ty were decreased when quercetin concentration was increased in the range of 0~100 μM quercetin and 37.5~300 μM L-tyrosine ( A ). Regression of two parameters derived two inversed inhibition constants, K I1 = 33.9 μM and K I2 = 417 μM, which means that quercetin is a highly competitive mixed inhibitor ( B , C ). Data were shown as averages and standard errors from triplicated samples. Table 1. Enzyme units of Bm Ty’s two activities at various pH conditions. Table 1. Enzyme units of Bm Ty’s two activities at various pH conditions. pH Enzyme Unit [U/μmol Enzyme] 1 Monophenolase Activity Diphenolase Activity 5 117 ± 6 202 ± 5 6 231 ± 19 494 ± 5 7 376 ± 19 699 ± 42 8 276 ± 23 879 ± 28 1 Values are shown as average ± standard deviation. MDPI and ACS Style Song, H.; Lee, P.-G.; Kim, H.; Lee, U.-J.; Lee, S.-H.; Kim, J.; Kim, B.-G. Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols. Microorganisms 2021, 9, 1866. https://doi.org/10.3390/microorganisms9091866 AMA Style Song H, Lee P-G, Kim H, Lee U-J, Lee S-H, Kim J, Kim B-G. Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols. Microorganisms. 2021; 9(9):1866. https://doi.org/10.3390/microorganisms9091866 Chicago/Turabian Style Song, Hanbit, Pyung-Gang Lee, Hyun Kim, Uk-Jae Lee, Sang-Hyuk Lee, Joonwon Kim, and Byung-Gee Kim. 2021. "Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols" Microorganisms9, no. 9: 1866. https://doi.org/10.3390/microorganisms9091866 Article Metrics Supplementary File 1: Song, H.; Lee, P.-G.; Kim, H.; Lee, U.-J.; Lee, S.-H.; Kim, J.; Kim, B.-G. Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols. Microorganisms 2021, 9, 1866. https://doi.org/10.3390/microorganisms9091866 Song H, Lee P-G, Kim H, Lee U-J, Lee S-H, Kim J, Kim B-G. Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols. Microorganisms. 2021; 9(9):1866. https://doi.org/10.3390/microorganisms9091866 Song, Hanbit, Pyung-Gang Lee, Hyun Kim, Uk-Jae Lee, Sang-Hyuk Lee, Joonwon Kim, and Byung-Gee Kim. 2021. "Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols" Microorganisms9, no. 9: 1866. https://doi.org/10.3390/microorganisms9091866	https://www.mdpi.com/2076-2607/9/9/1866/htm
Climate \| Free Full-Text \| The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development Climate changes are a phenomenon that can affect the daily activities of rural communities, with particular emphasis on those directly dependent on the agricultural and forestry sectors. In this way, the present work intends to analyse the impact that climate changes have on forest risk assessment, namely on how the occurrence of rural fires are affecting the management of the forest areas and how the occurrence of these fires has evolved in the near past. Thus, a comparative analysis of the data provided by IPMA (Portuguese Institute of the Sea and the Atmosphere), was carried out for the period from 2001 to 2017 with the climatic normal for the period between 1971 to 2000, for the variables of the average air temperature, and for the precipitation. In this comparative study, the average monthly values were considered and the months in which anomalies occurred were determined. Anomalies were considered in the months in which the average air temperature varied by 1 & deg;C than the value corresponding to the climatic norm, in at least 50% of the national territory. The same procedure was repeated for the variable precipitation, counting as anomaly the occurrence of a variation in precipitation of 50%, also in 50% of the national territory. Then the calculation of the moving averages for cycles of 3, 5 and 7 periods were applied, and the trend lines were projected. Subsequently, the relationship between the results obtained and the occurrence of rural fires as well as the spatial distribution of forest area, species and structure were analyzed. From the results obtained it was possible to confirm the existence of a tendency for the occurrence of climatic anomalies, highlighting the occurrence of an increasing number of months with temperatures higher by at least 1 & deg;C. It was possible to foresee the relation between the occurrence of rural fires and the periods of anomaly and absence of precipitation. From the results obtained it is also possible to infer that, analyzing the tendency for these phenomena to occur, it can be necessary to change the & ldquo;critical period of rural fires& rdquo;, since it is verified that what is currently in use does not covers the entire period where anomalies occur and where large-scale rural fires potentially can happen. The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development by Leonel J. R. Nunes 1,* , Catarina I. R. Meireles 1 , Carlos J. Pinto Gomes 1,2 and Nuno M. C. Almeida Ribeiro 1,3 Author to whom correspondence should be addressed. Climate 2019 , 7 (6), 78; https://doi.org/10.3390/cli7060078 Received: 18 April 2019 / Revised: 23 May 2019 / Accepted: 28 May 2019 / Published: 1 June 2019 (This article belongs to the Special Issue Climate Change, Carbon Budget and Energy Policy ) Abstract : Climate changes are a phenomenon that can affect the daily activities of rural communities, with particular emphasis on those directly dependent on the agricultural and forestry sectors. In this way, the present work intends to analyse the impact that climate changes have on forest risk assessment, namely on how the occurrence of rural fires are affecting the management of the forest areas and how the occurrence of these fires has evolved in the near past. Thus, a comparative analysis of the data provided by IPMA (Portuguese Institute of the Sea and the Atmosphere), was carried out for the period from 2001 to 2017 with the climatic normal for the period between 1971 to 2000, for the variables of the average air temperature, and for the precipitation. In this comparative study, the average monthly values were considered and the months in which anomalies occurred were determined. Anomalies were considered in the months in which the average air temperature varied by 1 °C than the value corresponding to the climatic norm, in at least 50% of the national territory. The same procedure was repeated for the variable precipitation, counting as anomaly the occurrence of a variation in precipitation of 50%, also in 50% of the national territory. Then the calculation of the moving averages for cycles of 3, 5 and 7 periods were applied, and the trend lines were projected. Subsequently, the relationship between the results obtained and the occurrence of rural fires as well as the spatial distribution of forest area, species and structure were analyzed. From the results obtained it was possible to confirm the existence of a tendency for the occurrence of climatic anomalies, highlighting the occurrence of an increasing number of months with temperatures higher by at least 1 °C. It was possible to foresee the relation between the occurrence of rural fires and the periods of anomaly and absence of precipitation. From the results obtained it is also possible to infer that, analyzing the tendency for these phenomena to occur, it can be necessary to change the “critical period of rural fires”, since it is verified that what is currently in use does not covers the entire period where anomalies occur and where large-scale rural fires potentially can happen. Keywords: climate changes ; Portuguese forest ; rural fires ; pests ; invasive species 1. Introduction Climate changes can be defined as global variations of the Earth’s climate, due to natural causes, but also to the action of Man [ 4 ]. Climate changes occurs at very different times and over all climatic parameters, such as temperature, precipitation, cloudiness, and so on. The designation “greenhouse effect” is applied to the phenomenon of retention of solar radiation by Earth’s atmosphere through a layer of gases called “greenhouse gases”. Without them life as it is known would not be possible, since the planet would be too cold [ 5 ]. Among these gases are carbon dioxide, nitrogen oxides and methane, which are produced mainly by industry, agriculture and the combustion of fossil fuels. The industrialized world contributed to increase the concentration of these gases by about 30% during the 20th century, when, without human action, nature was able to balance these emissions [ 6 ]. Today, there is a consensus about the idea that the current model of energy production and consumption is generating global climate changes, which in turn will cause serious impacts both on planet’s environment and on socioeconomic systems [ 7 ]. In the distant year of 2001, the Third Assessment Report of the Intergovernmental Panel on Climate changes (IPCC) highlighted the evidence provided by observations of physical and biological systems showing that regional changes in climate, more specifically the increases in temperatures, were affecting different systems in several parts of the planet [ 8 ]. The report definitively stated that there were mounting evidences of the existence of climate changes and its derived impacts. However, the temperature increased by about 0.6 °C during the 20th century [ 9 ]. More recently, in 2013, the IPCC Summary for Policy Makers stated that “man’s influence on the climate is obvious. This is evident from the growing concentration of greenhouse gases in the atmosphere, and it is extremely likely that human influence is the dominant cause of warming since mid-20th century. The continuous emission of greenhouse gases will cause further warming and changes in all climatic components of the planetary system. Limiting climate changes will require a substantial and sustainable reduction in the production and emission of greenhouse gases” [ 10 ]. Climate changes affects everyone on the planet indiscriminately. The potential impact is huge with predictions of lack of potable water, major changes in food production conditions and rising mortality rates due to floods, storms, droughts and heat waves [ 11 ]. Climate changes are not only an environmental phenomenon, but also have profound economic and social consequences. The poorest countries, which are least equipped to deal with rapid changes, will suffer the worst consequences. Extinction of animals and plants is expected as habitats will change so sharply that many species will not be able to adapt in time to survive. The World Health Organization (WHO) warned that the health of millions of people can be threatened by increased malaria, malnutrition and diseases [ 12 , 13 ]. Portugal, because of its geographical situation and socioeconomic characteristics, is very vulnerable to climate changes. As a consequence, even if there are uncertainties that do not allow the expected climate changes to be quantified with sufficient precision, the information validated so far is sufficient to take immediate action, in accordance with the so-called “Precautionary Principle”, which to is made reference in Article 3 of the United Nations Framework Convention on Climate Change (UNFCCC) [ 14 ]. Inertia, delays and the irreversibility of the climate system are very important factors to be taken into account and, the longer it takes to start action, more the effects of increasing concentrations of greenhouse gases will be less reversible [ 15 ]. Climate change presents major challenges for the Portuguese forestry sector. The effects of climate changes on forest ecosystems in Portugal are already evident in many respects. Anticipated impacts of future climate scenarios indicate a progressive intensification of these effects as the 21st century progresses, e.g., in the distribution of forest formations, structural and functional changes, in certain parameters of forest health, greater vulnerability to extreme weather events and rural fires, and a change in the flow of environmental goods and services that forests provide [ 16 ]. The interactions between the forest area and the problem of climate changes must be analyzed from two perspectives. On the one hand, it is necessary to contemplate what the forests can bring to the reduction of this problem, being a path to mitigation, and on the other hand, what impact climate changes can have on forests, analyzing their adaptation and evolution [ 17 ]. These interactions are not independent and are affected by complex interconnection and cause-effect processes. For example, the importance of forests to mitigate CO 2 concentrations can be affected if the impact of climate changes reduce their storage capacity through growth and development constraints, or increases the problem of rural fires. In other words, it is necessary to define and apply tools to manage forests more efficiently, to tackle the problem of climate changes, interlinking adaptation and mitigation, with a view to adaptation in order to mitigate its effects and consequences [ 18 , 19 ]. In Portugal, forests occupy approximately 3.2 million hectares, about 35% of the total area of the country [ 20 ]. Portuguese forests provide many benefits and services, including clean water and air, recreational and leisure spaces, wildlife habitats, carbon sequestration and storage, climate regulation, and a variety of forest products with a large impact on the economy [ 21 , 22 ]. Climate influences the structure and function of forest ecosystems and plays a key role in forest health. A changing climate can intensify many of the threats to forests, such as the outbreak of pest outbreaks, fires, drought and the very development of populations there [ 23 ]. Climate changes directly and indirectly affect the growth and productivity of forests through changes in temperature, precipitation, climate and other factors. In addition, high levels of carbon dioxide can also affect plant growth. These changes influence the complex forest ecosystems in various ways [ 24 ]. Together with the impacts resulting from the effects of climate changes, forests face impacts due to the development of land management, namely due to their use and occupation, periodic rural fires and atmospheric pollution. Although it is difficult to separate the effects of these different factors, the combined impact is already causing changes in Portuguese forests. As these changes are expected to continue in the coming decades, some of the economic aspects provided by forests may be compromised in the short term [ 25 ]. This review article is intended to approach climate changes, starting from the global perspective and then to deal specifically with the Portuguese situation, from the point of view of impacts. Following sections deal specifically with the evolution of the climate in Portugal, with the objective of understanding how these variations can influence the development of the forest. In this way, an analysis of the current state and the development of the climate in the last years is presented, both in terms of the evolution of the average air temperature and precipitation evolution. An analysis is also made of the effects of the changes and their relationship with the occurrence of rural fires in Portugal, with particular attention to the period 2001 to 2017, and to the way in which these occurrences interfere in the development and evolution of forests. Finally, an analysis is made of the impacts of climate changes and its consequences on Portuguese forests, in particular due to the increase in the occurrence of rural fires, pests and occurrence of forest invasive species. 2. Impact of Climate Changes in Forest Production 2.1. Forest Growth and Productivity There are several aspects related to climate changes that are most likely to significantly affect the growth and productivity of forest species. Examples of such factors are the rise in temperature, changes in precipitation levels and increases in the concentration of carbon dioxide [ 26 ]. The rise in temperature generally increases the duration of a plant’s growing season. This factor also contributes to the change in the geographic dispersion of some tree species. In this way, the habitats of some types of trees tend to move to the north or to higher altitudes. Other species will be at risk locally or regionally if the conditions in their current geographical ranges are not the most appropriate. For example, species that currently exist only on top of mountains in some regions may disappear as the climate warms up as these species cannot evolve to a higher altitude [ 27 ]. Climate change is most likely to increase the risk of drought in some areas and the risk of extreme rainfall and flooding in other regions [ 28 ]. Increased temperatures change the defrosting time, affecting the seasonal availability of water. Although many trees achieve up to a certain degree of drought, rising temperatures can make future droughts more damaging than they have in the past. In addition, drought increases the risk of rural fires, since dry trees and shrubs provide fuel for the fires. Drought also reduces sap trees’ ability to produce sap, which protects them from destructive insects and disease [ 29 ]. Carbon dioxide is needed for photosynthesis, the process by which green plants use sunlight to grow. With sufficient water and nutrients, increases in atmospheric CO 2 concentration may allow trees to have higher growth rates, which may alter the distribution of tree species. Growth will be higher on nutrient rich soils, with no water limitations, and will decrease with reduced fertility and water supply [ 30 ]. 2.2. Pests, Invasive Species and Rural Fires Climate changes can change the frequency and intensity of negative impacts on the forest, such as pest outbreaks, invasive species proliferation, rural fires and storm surges. These disturbances can reduce forest productivity and change the distribution of forest species. In some cases, forests may recover from a disturbance, but in other cases, existing species may evolve or disappear. In these cases, new plant species that colonize a given area create a new type of forest [ 31 ]. According to the opinion of several authors [ 32 , 33 , 34 ], with temperatures rising as the climate changes and warms, insects will become more active, and consequently more ravenous and abundant, increasing the likelihood of occurrence of insect pests. In this way damage to crops, both agricultural and forestry, is bound to increase [ 32 ]. This argument gains strength, as the increase in temperature accelerates the metabolism and reproduction of insects. The same authors estimate that each degree of increase in temperatures will mean increased damage in crops of 10% to 25% [ 32 ]. Unlike mammals and birds, insects heat or cool according to their environment. When an insect warms up, its metabolism accelerates. The faster you burn energy, the more voraciously the insect feeds and the sooner you can reproduce. The analysis of this information has allowed researchers to conclude that the growth rates of the populations are not very different among the different types of insects, allowing the development of a mathematical model that simulates the growth of the insect populations. This simulation allows us to infer the damage caused by these same populations of insects in the crops, both agricultural and forestry [ 32 , 34 ]. Tropical insects generally are already close to the upper limit of their temperature tolerance, so it can be concluded that it will not be in these regions that the greatest variations in insect populations will surely be observed. In areas with a more temperate climate, insects can significantly accelerate their activity, causing more damage to the crops. Variations in temperature may encourage or discourage insect species from invading new territories. Temperatures can also affect the parasites that attack the same insects that attack the crops, so the end result will greatly depend on the ability of all stakeholders to evolve and adapt to the new reality [ 32 , 35 ]. A number of biotic and abiotic agents have been identified in Portugal that are capable of causing physiological imbalances responsible for changes in tree development and that may be associated with the high frequency with which rural fires occur, which may lead to the onset of pests and diseases. Table 1 presents some of these pests that constitute major sanitary problems for the Portuguese forest. The xylophagous or subcortical insects that attack the trunk can be considered as the most serious pests, since they block the circulation of sap, putting at risk the survival of the tree. Some of these insects also have the ability to inoculate agents that contribute to the weakening and death of trees [ 36 ]. Insect attacks on leaves usually do not jeopardize tree survival (with the exception of very severe attacks on young stands), as the young tree has been completely stripped of needles, leading to the tree’s death. In other situations, for example with older and larger trees, canopy regeneration may occur, although it may show a decrease in its annual growth rate, since the energy reserves will be channeled for the renewal of the foliage [ 36 ]. This situation is more critical in the resinous species since its leaf surface is renewed in a slower way than in the hardwood species. However, it is the pests and diseases introduced in Portugal that cause the most damage to the forest. This is an example of the introduction of the woody pine nematode ( Bursaphelenchus xylophilus ) in 1999, which is currently the most serious forest health problem in Portugal, and is responsible for enormous economic losses, not only due to the dead pine trees, but also due to the strong restrictions on the export of pinewoods [ 36 ]. Eucalyptus also suffered from the introduction of exotic species, such as mortality caused by the eucalyptus borer ( Phoracantha semiounctata ) in the late 1990s, mainly in Beiras and Alentejo and, more recently, the damage caused by the defoliant weevil in the North and Center regions [ 36 ]. Recently, other exotic insects have been registered in Portugal, such as the eurasian golden-bug ( Thaumastocoris peregrinus ) and the chestnut-horned wasp ( Dryocosmus kuriphilus ) [ 37 ]. Its medium and long-term impact in the national territory is not yet known, but because of the risk of being an emerging pest, action plans have already been implemented for its control [ 36 ]. Lack of natural controls such as predators or pathogens, as well as the fact that tree defenses are unsuitable for these harmful agents, may allow insects to spread. Climate changes could contribute to an increase in the severity of future insect outbreaks. Rising temperatures may allow some species of insects to develop more rapidly, alter their seasonal life cycles, and expand their activity to other latitudes than usual [ 38 , 39 ]. In 2017, rural fires consumed more than 442,000 hectares of forest in Portugal, causing 104 human casualties and more than 600 million euros in losses [ 40 , 41 ]. High temperatures and drought conditions during the early summer contributed to this tragic scenario [ 42 ]. The rural fires of 2017 were the largest catastrophe in the country in terms of the number of fatalities since the fateful floods of 1967 [ 43 ]. Rural fires cause a feeling of impotence in the population, which considers them almost a fatality intrinsic to the Portuguese forest, to the point of generalizing the expression “fire season”, as if it were naturally part of the calendar as another season [ 44 ]. Since 2000, 200 people have died as a result of rural fires in Portugal [ 45 ]. As mentioned earlier, hundreds of thousands of hectares of forest have disappeared, accounting for more than half of the area burned in Europe in 2017 [ 46 ] and comprising a great historical landmark, since the fire decimated a significant part of the Pinhal do Rei in Leiria, from where the legend must have left the mythical wood that served to build the ships and caravels that led the Portuguese in the period of maritime expansion [ 44 ]. But it was not only the past that was destroyed, since the industrial and agricultural sectors were also largely hit, putting a high number of jobs at risk. However, this event is not new and in 2017 it only reached the contours that it reached due to the absurd number of deaths and injuries, since in previous years similar phenomena were observed, except for the number of victims. The effect of climate changes is expected to contribute to increasing the extent, intensity and frequency of rural fires in certain areas of the country. Warmer spring and summer temperatures, coupled with reduced availability of water, dry woody materials in forests and increase the risk of rural fires. Fires can also contribute to the very phenomenon of climate changes, as they can cause large and rapid releases of carbon dioxide into the atmosphere [ 47 ]. It is important to note that fire activity is not only determined by drought as a structural basis and by meteorology as the conjunctural basis of risk. The continued aridity has effects of greater temporal availability of all vegetation to burn, resulting in campaigns of continuous or extended fires, creating the conditions for large fires more easily and quickly than under the “normal” regime of situations [ 48 ]. The concept of “aridity” of fuel has been related in the USA with the increase in rural fires in size and severity [ 49 ]. This concept, as well that of fine fuel moisture content (FFMC) [ 50 ], is related to climate changes and contributes to the regime of extreme fires of the past gradually becoming the current normal fire regime [ 51 ]. Spring weather, and especially June weather conditions, led to very significant fuel “aridity” conditions. The months of June 2015–2017 along with the four-year period 2003–2006 were the hottest since record keeping, coinciding with years of larger fires [ 52 ]. There is, however, a significant difference between 2017 and the years 2003 to 2006, which means that in 2017 the concept of “aridity” of fuel has reached its maximum in Portugal by making a warm June to hot spring happen, what differentiates 2017 from the years of great fires of the recent past. As a result, in 2017 there was an advance of the first major fires of the year to June, when in the past they had always occurred in late July and early August. This change constitutes a clear impact of the effect of climate changes on contemporary fire regimes [ 53 ]. In the data presented in Table 2 it can be observed that the occurrence of rural fires has been a recurring situation in recent years. The table shows the area burned in fires from 1980 to 2017, as well as the number of ignitions occurred during the same period, in order to allow the correlation between the fires and the burning area and the annual costs caused by rural fires. From the analysis of the data it is possible to infer a tendency for the growth of the area burned in rural fires, mainly from the 90s, although this tendency has already occurred since the 80s, but with less significance. According to the Portuguese Nature and Forests Conservation Survey (ICNF) available data, the 10 years with the highest area burned all occurred after this period, including the peak year in 2017. This period coincides with many of the hottest years recorded throughout the country. This type of occurrence, especially when it occurs simultaneously, can interact with one another, or with changes in temperature variation and amount of precipitation, to increase the risks for the occurrence of several impacts on the forests. For example, drought can weaken trees and make the forest more susceptible to rural fires or outbreaks of insect pests and diseases. Similarly, after the occurrence of a forest fire, the forest may become more vulnerable to insect pests and diseases. This permanent occurrence of rural fires in Portugal, in addition to the environmental impact that is easily recognized, also has a very significant impact on the economic perspective. In fact, as already mentioned, the trivialization of the “fire season” as if it were a seasonally repeatable period of the Portuguese year led to the inclusion of significant amounts of costs in the state budget for the prevention of rural fires, in addition to the creation of an increasingly modern and prepared device to combat the fires. Table 2 presents as well the costs related to the occurrence of rural fires from 1980 to 2017. From the simple analysis of the data, there is a progressive increase in costs associated with the growth of the burned areas. The amount presented represents the sum corresponding to the amount spent each year on the prevention, combat and costs associated with the damages caused by rural fires. This phenomenon of rural fires in Portugal, which it is now beginning to be understood can be boosted by climate changes, does not however originate in these changes or even natural causes in the overwhelming majority of occurrences. Nowadays, the cause of the origin of the great majority of the rural fires is already investigated, being therefore possible to refute the thesis of the natural origin for these occurrences. Both the ICNF and the Nature and Environment Protection Survey (SEPNA), belonging to the National Republican Guard (GNR), have the human and technical capacity to analyze the causes that caused a certain ignition and disseminate the results of the investigations in documents that are available at their respective websites, which can be followed at the addresses, respectively ( www.icnf.pt and http://www.gnr.pt/atrib_SPENA.aspx ). From the analysis of these data we obtain the results that are presented in Table 3 . From the analysis of the data presented in Table 3 , it can be observed that in the vast majority of cases, rural fires have an accidental, negligent or willful origin, origin in human activity, and the role of natural phenomena to which responsibilities, are clearly very insignificant. This group includes, for example, the occurrence of dry thunderstorms, very frequent in the summer. However, as shown in the previous table, with a very insignificant weight in the overall number of occurrences. Among all the frequent causes, it continues to be the “use of fire”, mainly in the form of burnings to eliminate agricultural and forest residues, that is the main cause for the occurrence of rural fires ( Figure 1 ). 3. Evolution of Climate Changes in Portugal 3.1. Framework As previously mentioned, climate can be defined as the set of long-term meteorological conditions prevailing in a given area. Thus, in this perspective, the average values of the climatic variables of a given location will be more representative according to the time interval used in the analysis, thus constituting a time serie. In this way, the same results are not obtained when comparing a time interval of one year with one of 10 years, or one of 100 years. It is important to have long time series of data to analyze the variations and the evolution of the climate. For example, the Portuguese Institute of the Sea and the Atmosphere (IPMA), has available series of meteorological data dating back to 1865 [ 55 ]. The World Meteorological Organization (WMO) agreed that climate characterization is done by analyzing the mean values of the various climatic elements over a period of 30 years [ 56 , 57 ]. This period is the normal value of a climatic element and represents the average value corresponding to a sufficiently long number of years to be assumed to represent the predominant value of that element at the site under consideration [ 58 , 59 ]. Similarly, the WMO designates the statistical values obtained for periods of 30 years, starting in the first year of each set of years (1901–1930, 1931–1960, 1961–1990, 1991–2020) [ 60 ]. These are the reference normals or average representative values, however, can be calculated and used climatological normals based on intercalary periods, for example periods like 1951–1980 or 1971–2000 [ 55 ]. The Portuguese Institute of the Sea and Atmosphere (IPMA) provides online information on the climatological normal of 21 meteorological stations for the period 1971–2000, including monthly and annual values of the main climatic elements. In the same website are also available the average values of air temperature and total precipitation. For the two climatic parameters selected, average air temperature and precipitation, IPMA provides information on the occurrence of anomalies by creating maps of isolines representing, in the case of average air temperature, areas where the air temperature exceeded (positive or negative) the values of the last climatic normal (period from 1971 to 2000). Likewise, for precipitation, isoline maps are also presented, thus representing percentage of the amount of precipitation occurred compared to normal climatic conditions (period 1971 to 2000). Subsequently, new maps of isolines based on the previous ones, made available by the IPMA website, were constructed, which were simplified in order to facilitate the reading and counting of the number of anomalies that occurred, both for the average air temperature and for the precipitation. After counting the anomalies in the period from 2001 to 2017 for the two parameters selected, tables were elaborated presenting the data. For the verification of the existence of a trend of occurrence of events and to allow a better visualization of the results, a moving average model was applied, because this method facilitate the smoothing of the plotting of the data allowing an easier visualization of the eventual trend. 3.2. Air Temperature Anomalies The most well-known and referred parameter when it comes to the subject of climate changes is surely the rise of air temperature. If on the one hand the scientific community addresses other parameters with the same concern and capacity for analysis, civil society refers to this particular issue, often without understanding its real effects, but mainly because it is the most approached by the media. It is a rare day when no news comes out in all kinds of media that do not allude to “global warming” and the “greenhouse effect”, or very specifically to its effects and consequences anywhere in the world, such as occurrences of rural fires, hurricanes, floods, long periods of drought, rising sea levels or changing monsoon cycles. Thus, this section analyzes the occurrence of anomalies in the average air temperature. In this particular case, the occurrence of an anomaly is considered whenever the average monthly temperature exceeds 1 °C, compared to normal climatic conditions during the period 1971–2000. Based on this analysis, isoline maps were constructed, as the presented in the example of Figure 2 , which visually indicate which regions of the country have exceeded +1 °C (colored in red), −1 °C (colored in blue) and which were similar to normal climatic (colored in green). An abnormal month is considered when at least 50% of the national territory has been subjected to temperature values above or below 1 °C compared to normal climatic conditions (period 1971–2000). Table 4 shows the anomalies observed in the period between 2001 and 2017. As can be seen, there was a significant set of anomalies in all the constituent years of the period, with a maximum of anomalies of 7 occurring in 2017 2005, 2006, 2009, 2015 and 2017. The lowest number of anomalies was reached in 2004 and 2007, with 3 occurrences. Based on these data, it is necessary to determine the existence of a tendency for an increasing number of anomalies, that is, to determine if the number of anomalies occurring in the period between 2001 and 2017 shows a tendency to occur in some sense. For this inquiry the simple moving average method was used [ 61 ]. The determination of the simple moving average of a set of n elements is obtained by calculating the unweighted averages of the subsets of n elements in a given set of data. For example, given a set of n elements p 1 , …, p n , the first element of the moving average is given by Equation (1): M n = p 1 + … + p n n = 1 n (1) the second is given by Equation (2): M n ′ = p 2 + … + p n + 1 n = 1 n ∑ i = 2 n + 1 p i (2) or even by Equation (3): M n ′ = M n + p n + 1 n − p 1 n (3) and so on until p n−n+1 ,…,p n . The same Table 5 presents the results of the application of the simple moving average method and in Figure 3 the trend lines are presented. In this particular situation the method was applied for 3, 5 and 7 periods, since it was understood that for the number of data available, it would be sufficient. The application of the periods consists in the aggregation of data groups according to the period used, and in the performance of the average of that group, the result obtained corresponds to the average element of the group. For example, for an average of three periods, the first three years, 2001, 2002 and 2003 are selected, and the average number of occurrences for these three years are determined. The final result is assigned to the middle element, in this case it is the year 2002. Then the operation was repeated for the group of the next three elements, 2002, 2003 and 2004, and so on. For the calculation of the means of five and seven periods, the procedure is similar, but now, instead of selecting three elements, five or seven will be selected, repeating the operation for all the elements of the sample. The higher the number of data available, more averages with different periods can be calculated, the data being analyzed as linearly as possible. It is in this way that one can determine if there is a growing or decreasing tendency of the occurrence of a given event ( Figure 2 ). In the previous figure is showed the projection of the data previously presented in Table 1 . As can be seen, the real data or the total occurrences, represented by the blue line, allow the creation of a trend line and indicate by itself a perspective increase in the number of occurrences. However, with the application of the simple moving averages method, it is verified that the lines corresponding to each of the averages, the average of three periods being represented by the brown line, the average of the five periods represented by the green line and the average of seven periods represented by the purple line, indicate in a much more visible way an increasing tendency for the occurrence of climatic anomalies. This fact is even more noticeable when one observes the lines or lines of trend, which clearly indicate an increasing trend towards a more frequent occurrence of this event. In addition to this verification of the tendency for a given event to occur, in this case the occurrence of climatic anomalies, it is also necessary to verify the probability of occurrences occurring in one month to the detriment of another, since this seasonality, associated to the occurrence of precipitation and to the biological cycles of plant growth may be determinant for the increased risk of occurrence of rural fires, as well as for their degree of intensity and severity. Table 5 presents the distribution of climatic anomalies by the months of the constituent years of the period under analysis. As can be seen in the data presented in Table 5 , although there is a dispersion for all the months of the year, since climatic anomalies occurred in all months of the year without exception. Over the last 17 years under analysis in this study, there is a higher concentration of these events in the spring and early summer months (April, May and June), so if factors are also anomalous in the following months, summer and autumn, also associated to the occurrence of precipitation anomalies, a strong probability of occurrence of ideal conditions for the outbreak of rural fires of great intensity and severity. As can be seen, there is also a strong tendency for air temperature anomalies to occur during the summer and autumn months, so it can be said that the probability of repeating situations such as those occurring in 2017, favorable for the outbreak of fires in summer and autumn, is very high. 3.3. Precipitation Anomalies Similar to the methodology used for the previous section, maps of isolines were created on the maps made available on the IPMA website ( www.ipma.pt ). In these maps, the zones of the country where precipitation was at least equal to that of the normal climatic period used in this study, the period 1971 to 2000 were defined as an anomaly when in a given month, in at least 50% of the continental national territory, precipitation was lower than that occurred in the period of normal climatic conditions. Table 6 shows the precipitation anomalies observed in the period between 2001 and 2017. As can be seen, there was a significant set of anomalies in all the constituent years of the period, with a maximum of 11 anomalies occurring until 2017, in the years of 2015 and 2017, but in 2008 there were 10 anomalies, and in the years 2004, 2007, 2009 and 2012, there were 9 anomalies. The lowest number of anomalies was reached in the years 2006 and 2014, with 5 occurrences. Table 6 presents the results of the application as well of the simple moving average method and in Figure 4 the trend lines are presented, using the same methodology described in the previous section. Also, in this situation the method was applied for 3, 5 and 7 periods, since it was understood that for the number of data available, it would be sufficient. Based on these results the graphic presented in Figure 4 was constructed, where the trend lines are also projected, and the projection of data previously presented in Table 3 are shown. As can be seen, the actual data or total counted occurrences, represented by the blue line, allow the creation of a trend line and indicate in itself a growth perspective of the number of occurrences. However, with the application of the simple moving averages method, it is verified that the lines corresponding to each of the averages, the average of three periods being represented by the red line, the average of the five periods represented by the green line and the average of seven periods represented by the purple line, indicate in a much more visible way an increasing tendency for the occurrence of climatic anomalies. This fact is even more noticeable when trend lines are analyzed, which clearly indicate an increasing trend towards a more frequent occurrence of this event, similar to what had already happened in the previous section, in the analysis of air temperature anomalies. As can be seen in Table 7 , there is a dispersion of the occurrence of anomalies for all the months of the year, since occurred climatic anomalies in all months of the year without exception. Over the last 17 years under analysis in this study, there is a greater concentration of these events in the spring and early summer months (April, May and June), so if factors are also anomalous in the following months, summer and autumn, also associated with the occurrence of precipitation anomalies, there is a strong probability of occurrence of ideal conditions for the outbreak of rural fires of great intensity and severity. It also appears that the occurrence of periods with precipitation at levels lower than usual would occur more and more frequently and can be said that the trend of the period between 2001 and 2017 is the repetition of periods of low precipitation. As can be observed, there is also a certain tendency for precipitation anomalies to occur during the spring and summer months, so it can be said that the probability of repeating situations such as those occurring in 2017, with very favorable conditions for the outbreak of rural fires in summer and autumn is very high, especially when the conditions previously discussed are combined. However, these considerations still have to be validated through the analysis of a longer time period, since the use of a range composed of only 17 years seems manifestly short so that definitive conclusions can be drawn on the evolutionary tendency of the climate. Several authors indicate in their work that a period of less than 30 years may not be representative to justify an evolutionary analysis of the climate [ 62 , 63 , 64 ]. Many point to the analysis of time intervals of 30, 50, or even more years. However, there are other authors who point to the use of more restricted time windows, in order to allow the analysis of short duration sensitivity, which may somehow indicate trends in a given direction, for one or more variables [ 4 , 65 ]. Notwithstanding the fact that this is not the most appropriate conclusion, in order to verify if there is an effective trend of climate change, the data indicate a tendency for an increasing number of climate anomalies, both associated with monthly air temperature average, and to the monthly average precipitation, when compared to the normal period 1971–2000. A good possibility for an expedited confirmation of the existence of a trend in time series is through the use of the non-parametric Mann–Kendall test, suggested by the WMO to evaluate the trend in time series of environmental data. The non-parametric Mann–Kendall test is commonly employed to detect monotonic trends in series of environmental data, climate data or hydrological data [ 66 ]. The null hypothesis, H 0 , is that the data come from a population with independent realizations and are identically distributed. The alternative hypothesis, H A , is that the data follow a monotonic trend. The Mann–Kendall test statistic is calculated according to: S = ∑ k = 1 n − 1 ∑ j = k + 1 n s g n ( X j − X k ) (4) where, s g n ( x ) = { 1 i f x > 0 0 i f x = 0 − 1 i f x < 0 (5) The mean of S is E[S] = 0 and the variance, σ 2 is: σ 2 = { n ( n − 1 ) ( 2 n + 5 ) − ∑ j = 1 p t j ( t j − 1 ) ( 2 t j + 5 ) } / 18 (6) where p is the number of the tied groups in the data set and t j is the number of data points in the j th tied group. The statistic S is approximately normal distributed provided that the following Z-transformation is employed: Z = { S − 1 σ i f S > 0 0 i f S = 0 S + 1 σ i f S < 0 (7) The statistic S is closely related to Kendall’s τ as given by: τ = S D (8) where, D = [ 1 2 n ( n − 1 ) − 1 2 ∑ j = 1 p t j ( t j − 1 From the application of the Mann–Kendall test to the counted data of the anomalies occurring for mean air temperature and precipitation, summarized in Table 8 and in Figure 5 and Figure 6 , it can be inferred that there is in fact an increasing tendency for occurrence of this type of anomalies. Considering the hypothesis H0: there is no trend in the series, and the hypothesis Ha: there is a positive trend in the series, and since the calculated p-value is lower than the level of significance, we reject the null hypothesis H0 in favor of the alternative hypothesis Ha. 4. Discussion and Conclusions As stated previously, temperatures on Earth are suitable for life thanks to a natural process called the “greenhouse effect”. When solar radiation reaches the atmosphere, part of it is reflected into space, and part of it passes and is absorbed by the Earth. This causes the Earth’s surface to heat up. The heat is radiated out and absorbed by the gases present in the Earth’s atmosphere, the so-called “greenhouse gases” (GHG) [ 67 ]. This process prevents the heat from disappearing, causing the temperature to rise to +14°C instead of −19°C. There are many greenhouse gases responsible for additional warming of the atmosphere, which are produced in different manners. Most come from the combustion of fossil fuels in cars, factories and the production of electricity. The gas responsible for most of the heating is carbon dioxide. Other gases that contribute to heating are methane, which is expelled by landfills and agriculture (especially from the digestive systems of large animals in intensive production), nitrous oxide from fertilizers, gases used for cooling in industrial processes and massive loss of forest area, otherwise they would store CO 2 [ 68 , 69 ]. Forests, which function as warehouses for greenhouse gases, help to mitigate the effects of climate change. However, the biological diversity of forests is also directly and indirectly affected by changing climatic conditions. These changes question the extent to which forests could continue to sequester greenhouse gases in the future [ 70 ]. The models that represent the ecosystems and their variations in the different climatic scenarios suggest that the changes will present a variety of impacts on the distribution of the forest populations, as well as on the impact on the function and composition of the ecosystems. In general, habitats are expected to move towards the poles and progress in altitude, conquering new territories [ 71 ]. With the change of the habitats, forest biodiversity will be forced to adapt and as a result, species composition in forests will likely change, and species and populations that are already vulnerable will become potentially extinct. In addition, with climate changes, there will be a greater incidence of extreme weather events, such as floods and droughts. These types of events will further affect forest populations and may make forests more prone to disturbances such as fires, invasive species, diseases and pests [ 72 ]. A mixed and preferably autochthonous forest stand, consisting of several different tree species with different ecological requirements and the ability to adapt to the expected changes in temperature, precipitation, frequency of storms and pests, will allow for continuous adjustments according to the climatic evolution [ 73 ]. The problem of climate changes has been addressed in a continuous way in Portugal, both by elements of the academic world, which are drawing attention to the causes and consequences of the phenomenon, but also by other sectors of civil society, in particular by the political sector, which has since some time initiated an ambitious program for the implementation of measures aimed at minimizing the negative impacts of climate changes in the country. Although they are still are taking the first steps in understanding the causes and consequences, it is assumed by the national and international scientific community that countries with Mediterranean climate characteristics may be the most affected by climate changes [ 16 , 74 , 75 ]. In this sense, a significant effort has been made in Portugal to implement measures that contribute to mitigating the harmful effects of climate changes, which at least begin to have international recognition. On 18 June 2018, TSF radio station on its website published a news item entitled “Portugal is second in a ranking on ambition in goals and measures to comply with the Paris Agreement on climate changes, being only exceeded by Sweden. The vast majority of the Member States of the European Union (EU) are failing to reach the targets of the Paris Agreement, and Portugal is among the few countries that have appealed to goals and policies more ambitious in the area of energy and climate, such as reducing greenhouse gas emissions”. This information is the result of a study entitled “Off target: Ranking of European Union (EU) countries ‘ambition and progress’ in fighting climate changes”, which determines member states’ commitment to achieving energy policy and energy targets and the progress they are making in reducing greenhouse gas emissions and implementing programs for the use of energy from renewable sources and energy efficiency [ 76 ]. The planet’s climate has undergone major changes for several decades. The IPCC report indicates that climate warming is evident and that most is probably due to the increase in GHG concentrations caused by human activities, such as the widespread use of fuels, the decomposition of urban or livestock waste and the changes in the occupation of the soil. There is already irrefutable proof of this change. The temperature of the atmosphere at surface level has undergone a progressive warming from the beginning of the industrial era to the present day of approximately 0.6°C on average, with an even greater increase in some areas such as the poles or the Mediterranean region. The hottest years since recordings have occurred since 1990, as well as major seasonal changes, such as the decline of icy surfaces, rising sea levels, changes in the overall circulation flow of marine currents, and so on [ 77 , 78 , 79 ]. The frequency and severity of extreme weather events has increased. There is a more frequent occurrence of floods, heat and cold waves and periods of prolonged drought. An example of this is the news and constant warnings of hurricanes and storms of extreme force, which always cause high economic and personal damages. There is a total consensus on the part of the scientific community when it comes to attributing to the increased concentration of GHG generated by human activities the greater responsibility for the phenomenon of climate changes. The reality is that without the natural presence of some of these gases in the atmosphere, such as water vapor and CO 2 , creating the known greenhouse effect, Earth would be a very different place from what is known today, with average temperatures well below the current. Since the beginning of the industrial revolution, when large quantities of fossil fuels began to be burned to meet the energy needs of industrial processes, so far, the amount of CO 2 in the atmosphere has increased progressively. Likewise, other anthropogenic GHGs also increased their concentration in the atmosphere considerably. Demographic growth and the current socio-economic model put great pressure on the self-regulating capacity of the atmosphere, which is leading to a situation close to its limits and, according to some scientists, likely to overcome them. The main causes for GHG emissions vary according to the regions of the planet. Thus, in the northern hemisphere, the main causes are associated with energy production, industrial production and transportation, while in the southern hemisphere the main causes are associated with the change in land use, namely through the conversion of extensive forest areas into agricultural land or pastures. It should be noted that in recent years there was an effort in the industrialized countries, with some success in some cases like Portugal, for the reduction in carbon dioxide emissions. The reasons for this reduction are the introduction of more efficient technologies, the use of renewable energies, the increasing weight of the services sector and the relocation of the most polluting companies to less developed countries. However, the steady growth of these industrialized economies, as well as the significant increase in emissions in other sectors, such as transport and the domestic sector, have made the total amount of GHG emissions of human origin increase considerably in recent years. Forests like all other natural ecosystems are as susceptible to climate changes as other sectors (such as agriculture, for example, which are also highly vulnerable to climate and environmental changes). Unlike other sectors, where financial resources and technology can directly contribute to increasing the adaptive capacity of affected systems, natural forests depend on their own natural ability to adapt. Adding to all this human pressure, pressure for development, and climate changes itself, it is highly likely that the ability of forestry systems to adapt to the new situation quickly and efficiently will be exceeded. It is expected that different forest systems will have different sensitivities to changes in climate. Therefore, it is important to take into account that the conservation of forests for other uses, and their ability to sequester carbon dioxide, can contribute predominantly to carbon dioxide emissions in the future if forest systems are affected by natural or human influences. The way ecosystems respond to climate changes is usually guided by two paradigms: evolution and adaptation, as seen in previous sections. In the first one it is assumed that there will be a migration of ecosystems to other regions, almost intact, just looking for new locations where climatic and environmental conditions reproduce those where they currently are. The second paradigm assumes that as the climate and other factors change, ecosystems will change in the same location where they are today, which will interfere both in the variety of species and in their position in the ecosystem. In addition to the intrinsic value of natural ecosystems, ecosystems of all kinds, from the most natural to the most intensively managed, offer a variety of benefits to society as a whole. Some of the products originating from these ecosystems enter the market and contribute directly to economic development. For example, forests are a source of raw materials for a number of industries, such as the production of biomass pellets, pulp production, the production of wood pellets and the production of furniture. Forest ecosystems also provide a number of benefits to society, such as their role in regulating water flows, preventing erosion, maintaining biodiversity and temporary storage of carbon, which can be as long as the longer the forest species and the more extensive the forestry operation recommended, for example, in the Portuguese case, using native species such as oaks, holm oaks or cork oaks. Changes in soil cover caused by climate changes can have a number of impacts on these benefits, such as the ability of these systems to stabilize the landscape against erosion or sequester carbon dioxide. Even in regions where the amount of existing vegetation is expected to increase as a result of higher precipitation rates and increased growth due to the higher concentration of atmospheric carbon dioxide it can lead to an increase in the frequency and intensity of fires during a longer summer period. The increase in the occurrence of fires is already a threat not only to the vegetation cover, but also to the residential structures that are built in the rural areas, which are increasingly vulnerable. For this reason, it is very plausible that the changes caused in the natural ecosystems by the changes of the climate affect this set of benefits usually associated with the forestry activity. As seen in previous sections, burning, or simply the “use of fire”, are the main causes of the occurrence of rural fires in Portugal, and its control and, if necessary, prohibition is urgently needed. Although it is an ancestral practice, with the worsening of climatic conditions, especially during the summer period, but which, as previously seen, now also extended to spring and autumn, the risk associated with this practice has grown exponentially. Most likely, the need to extend the so-called “rural fires season” will arise in the short term, where all means of combat are on alert so that they can react in a timely manner to any emergency. Almost all climate models anticipate a decrease in the amount of precipitation in various parts of the globe, but they emphasize in particular the effects on the Mediterranean climate regions. The parallel effect of temperature increasing and reduction of precipitation may lead to a significant decrease in the amount of soil moisture. According to the information provided in the previous sections, there is already a tendency for climatic anomalies related to the increase of average air temperature in Portugal, associated with the occurrence of anomalies related to the amount of precipitation in several months of the year. That is, the trend indicates a strong probability of occurrence of periods of temperature rise associated with lower levels of precipitation. This lack can make trees more fragile from insect pest attacks and diseases, but also from increasing the likelihood of rural fires. The frequency and intensity of these occurrences will determine the type and rate of conversion of soil cover to a new state, for example the replacement of a forest composed of native species, by another one composed of invasive species. In Portugal, infestations of extensive forest areas by species of the genus Acacia begin to be very frequent, but there are others, such as Hakea , that begin to occupy very significant areas and are no longer limited to occupying space along the circulation ways. However, as a consequence of climate changes, forests can undergo more rapid changes; for example, unless a significant increase in precipitation occurs, the severity of rural fires may increase. For this reason, it is urgent to create a model of forest management that takes into account the phenomenon of climate changes and all associated variables, and not to forget that forests are a natural resource that can should be profitable, with a view to sustainability for the future. Author Contributions Conceptualization, L.J.R.N., C.I.R.M., C.J.P.G. and N.M.C.A.R.; methodology, L.J.R.N.; validation, C.I.R.M., C.J.P.G. and N.M.C.A.R.; writing—original draft preparation, L.J.R.N.; writing—review and editing, C.I.R.M., C.J.P.G. and N.M.C.A.R.; supervision, C.I.R.M., C.J.P.G. and N.M.C.A.R. Funding This research received no external funding. Acknowledgments Authors declare no need of further acknowledgements. Conflicts of Interest The authors declare no conflict of interest. References Füssel, H.-M. Vulnerability: A generally applicable conceptual framework for climate change research. Glob. Environ. Chang. 2007 , 17 , 155–167. [ Google Scholar ] [ CrossRef ] Kurten, B. 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Panoramic picture taken on 12/5/2017 from ESA—Ponte de Lima School of Agriculture of the Polytechnic Institute of Viana do Castelo (north Portugal), where it is possible to observe 9 fires in a rural environment occurring simultaneously. Figure 1. Panoramic picture taken on 12/5/2017 from ESA—Ponte de Lima School of Agriculture of the Polytechnic Institute of Viana do Castelo (north Portugal), where it is possible to observe 9 fires in a rural environment occurring simultaneously. Figure 2. Example of the creation of isolines maps for the anomalies of in February in the year 2019. In the left part is the image obtained at the Portuguese Institute of the Sea and the Atmosphere (IPMA) website and in the right side is the image created from the first one. Figure 2. Example of the creation of isolines maps for the anomalies of in February in the year 2019. In the left part is the image obtained at the Portuguese Institute of the Sea and the Atmosphere (IPMA) website and in the right side is the image created from the first one. Figure 3. Trend lines. As can be seen in the figures, the lines indicate an increasing tendency for anomalies to occur, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Figure 3. Trend lines. As can be seen in the figures, the lines indicate an increasing tendency for anomalies to occur, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Figure 4. Trend lines. As can be seen in the figures, the lines indicate an increasing tendency for anomalies to occur, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Figure 4. Trend lines. As can be seen in the figures, the lines indicate an increasing tendency for anomalies to occur, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Figure 5. p -values. Figure 6. Sen’s slope indicating the increasing probability for the occurrence of anomalies. As can be seen in the figures, the Sen’s slope indicates an increasing trend for the occurrence of anomalies, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Figure 6. Sen’s slope indicating the increasing probability for the occurrence of anomalies. As can be seen in the figures, the Sen’s slope indicates an increasing trend for the occurrence of anomalies, allowing it to be inferred that, over the years, an increasing number of anomalies can be expected to occur. Table 1. Main pests of the forest in Portugal (adapted from [ 36 ]). Table 1. Main pests of the forest in Portugal (adapted from [ 36 ]). Forestry Species Scientific Name of the Pest Eucaliptus Gonipterus platensis Phoracantha semipunctata Cork oak and holm oak Lymantria dispar Periclista spp. Tortrix viridana Curculio elephas Cydia splendana Coroebus undatus Coroebus florentinus Platypus cylindrus Xyleborus spp. Maritime pine and stone pine Thaumetopoea pityocampa Pineus pini Cinara maritima Leucaspis spp. Pissodes validirostris Dioryctria mendacella Leptoglossus occidentalis Orthotomicus erosus Tomicus spp. Ips sexdentatus Dioryctria sylvestrella Pissodes castaneus Monochamus galloprovincialis Table 2. Annual costs associated with rural fires (adapted from [ 20 ] and [ 54 ]). Table 2. Annual costs associated with rural fires (adapted from [ 20 ] and [ 54 ]). Year Nr. of Occurrences Burned Area (m 2 ) Total Costs (€) 1980 2349 44,251 €96,024,670 1981 6730 89,798 €194,861,660 1982 3626 39,556 €85,836,520 1983 4542 47,811 €103,749,870 1984 7356 52,710 €114,380,700 1985 8441 146,254 €317,371,180 1986 5036 89,522 €194,262,740 1987 7705 76,268 €165,501,560 1988 6131 22,434 €48,681,780 1989 21,896 126,237 €273,934,290 1990 10,745 137,252 €297,836,840 1991 14,327 182,486 €395,994,620 1992 14,954 57,012 €123,716,040 1993 16,101 49,963 €108,419,710 1994 19,983 77,323 €167,790,910 1995 34,116 169,612 €368,058,040 1996 28,626 88,857 €192,819,690 1997 23,497 30,535 €66,260,950 1998 34,675 158,369 €343,660,730 1999 25,473 70,613 €153,230,210 2000 34,107 159,605 €433,000,000 2001 26,947 112,312 €319,000,000 2002 26,576 124,619 €394,000,000 2003 26,219 425,839 €1,303,000,000 2004 22,165 130,108 €402,000,000 2005 35,824 339,089 €756,746,827 2006 20,444 76,058 €132,001,898 2007 20,316 32,595 €37,109,004 2008 14,930 17,565 €22,371,685 2009 26,136 87,421 €86,259,214 2010 22,026 133,091 €183,911,947 2011 25,222 73,829 €80,557,921 2012 21,179 110,232 €196,227,660 2013 19,294 152,690 €208,337,840 2014 7067 19,930 €27,503,169 2015 15,851 64,412 €119,406,200 2016 13,079 160,490 €460,000,000 2017 16,981 442,418 €616,000,000 Total 4,419,166 €9,589,826,075 Table 3. Causes of rural fires investigated by National Republican Guard/Nature and Environment Protection Survey (GNR/SEPNA) in 2016 (adapted from [ 20 ]). Table 3. Causes of rural fires investigated by National Republican Guard/Nature and Environment Protection Survey (GNR/SEPNA) in 2016 (adapted from [ 20 ]). Causes Percentage of Total Occurrences Undetermined 34.7% Use of fire 25.2% Arson 21.6% Re-ignition of rural fires 13.6% Accidental 3.5% Natural 0.7% Structural 0.6% Table 4. Results obtained from the application of the simple moving average method. Table 4. Results obtained from the application of the simple moving average method. Table 5. Distribution of climatic anomalies by the months of the constituent years of the period under analysis. Table 5. Distribution of climatic anomalies by the months of the constituent years of the period under analysis. Year J F M A M J J A S O N D 2001 a a a a a 2002 a a a a a 2003 a a a a a 2004 a a a 2005 a a a a a a a 2006 a a a a a a a 2007 a a a 2008 a a a a 2009 a a a a a a a 2010 a a a a a 2011 a a a a a 2012 a a a a 2013 a a a a a 2014 a a a a a a 2015 a a a a a a a 2016 a a a a a a 2017 a a a a a a a Total 7 6 6 10 10 12 8 8 9 9 4 2 Table 6. Results obtained from the application of the simple moving average method. Table 6. Results obtained from the application of the simple moving average method. Year Nr. of Occurrences 3 Periods Average 5 Periods Average 7 Periods Average 2001 6 2002 7 6 2003 6 7 7 2004 9 8 7 7 2005 8 7 7 8 2006 5 7 8 8 2007 9 8 8 8 2008 10 9 8 8 2009 9 8 8 8 2010 6 7 8 8 2011 7 7 8 8 2012 9 8 7 8 2013 8 7 8 8 2014 5 8 8 8 2015 11 8 8 2016 7 10 2017 11 Table 7. Distribution of climatic anomalies by the months of the years of the period under analysis. Table 7. Distribution of climatic anomalies by the months of the years of the period under analysis. Year J F M A M J J A S O N D 2001 a a a a a a 2002 a a a a a a a 2003 a a a a a a 2004 a a a a a a a a a 2005 a a a a a a a a 2006 a a a a a 2007 a a a a a a a a a 2008 a a a a a a a a a a 2009 a a a a a a a a a 2010 a a a a a a 2011 a a a a a a a 2012 a a a a a a a a a 2013 a a a a a a a a 2014 a a a a a 2015 a a a a a a a a a a a 2016 a a a a a a a 2017 a a a a a a a a a a a Total 11 12 9 11 12 13 15 11 10 6 9 14 Table 8. Summary of data obtained from the Mann–Kendall test. Table 8. Summary of data obtained from the Mann–Kendall test. Series/Test Kendall’s τ p -value Sen’s slope Temperature anomalies 0.281 0.144 0.077 Precipitation anomalies 0.242 0.194 0.148 MDPI and ACS Style Nunes, L.J.R.; Meireles, C.I.R.; Pinto Gomes, C.J.; Almeida Ribeiro, N.M.C. The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development. Climate 2019, 7, 78. https://doi.org/10.3390/cli7060078 AMA Style Nunes LJR, Meireles CIR, Pinto Gomes CJ, Almeida Ribeiro NMC. The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development. Climate. 2019; 7(6):78. https://doi.org/10.3390/cli7060078 Chicago/Turabian Style Nunes, Leonel J. R., Catarina I. R. Meireles, Carlos J. Pinto Gomes, and Nuno M. C. Almeida Ribeiro. 2019. "The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development" Climate7, no. 6: 78. https://doi.org/10.3390/cli7060078 Nunes, L.J.R.; Meireles, C.I.R.; Pinto Gomes, C.J.; Almeida Ribeiro, N.M.C. The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development. Climate 2019, 7, 78. https://doi.org/10.3390/cli7060078 Nunes LJR, Meireles CIR, Pinto Gomes CJ, Almeida Ribeiro NMC. The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development. Climate. 2019; 7(6):78. https://doi.org/10.3390/cli7060078 Chicago/Turabian Style Nunes, Leonel J. R., Catarina I. R. Meireles, Carlos J. Pinto Gomes, and Nuno M. C. Almeida Ribeiro. 2019. "The Evolution of Climate Changes in Portugal: Determination of Trend Series and Its Impact on Forest Development" Climate7, no. 6: 78. https://doi.org/10.3390/cli7060078	https://www.mdpi.com/2225-1154/7/6/78
Atmosphere \| Free Full-Text \| The Influence of Graphene Oxide on Nanoparticle Emissions during Drilling of Graphene/Epoxy Carbon-Fiber Reinforced Engineered Nanomaterials Graphene oxide (GO) nanoparticles are increasingly being used to tailor industrial composites. However, despite the advantages, GO has shown conceivable health risks and toxicity to humans and the environment if released. This study investigates the influence that GO concentrations have on nanoparticle emissions from epoxy-reinforced carbon fiber hybrid composites (EP/CF) during a lifecycle scenario, that is, a drilling process. The mechanical properties are investigated and an automated drilling methodology in which the background noise is eliminated is used for the nanoparticle emissions measurements. Real-time measurements are collected using a condensation particle counter (CPC), a scanning mobility particle sizer spectrometer (SMPS), a real-time fast mobility particle spectrometer (DMS50) and post-test analytical methods. The results observe that all three nanoparticle reinforced samples demonstrated a statistically significant difference of up to a 243% increase in mean peak particle number concentration in comparison to the EP/CF sample. The results offer a novel set of data comparing the nanoparticle release of GO with varying filler weight concentration and correlating it the mechanical influence of the fillers. The results show that the release characteristics and the influence in particle number concentration are primarily dependent on the matrix brittleness and not necessarily the filler weight concentration within the nanocomposite. The Influence of Graphene Oxide on Nanoparticle Emissions during Drilling of Graphene/Epoxy Carbon-Fiber Reinforced Engineered Nanomaterials by Kristof Starost and James Njuguna * Centre for Advanced Engineering Materials, School of Engineering, Robert Gordon University, Aberdeen AB10 7GJ, UK Author to whom correspondence should be addressed. Atmosphere 2020 , 11 (6), 573; https://doi.org/10.3390/atmos11060573 Received: 25 April 2020 / Revised: 18 May 2020 / Accepted: 26 May 2020 / Published: 1 June 2020 (This article belongs to the Special Issue Emissions, Transport and Fate of Pollutants in the Atmosphere ) Graphene oxide (GO) nanoparticles are increasingly being used to tailor industrial composites. However, despite the advantages, GO has shown conceivable health risks and toxicity to humans and the environment if released. This study investigates the influence that GO concentrations have on nanoparticle emissions from epoxy-reinforced carbon fiber hybrid composites (EP/CF) during a lifecycle scenario, that is, a drilling process. The mechanical properties are investigated and an automated drilling methodology in which the background noise is eliminated is used for the nanoparticle emissions measurements. Real-time measurements are collected using a condensation particle counter (CPC), a scanning mobility particle sizer spectrometer (SMPS), a real-time fast mobility particle spectrometer (DMS50) and post-test analytical methods. The results observe that all three nanoparticle reinforced samples demonstrated a statistically significant difference of up to a 243% increase in mean peak particle number concentration in comparison to the EP/CF sample. The results offer a novel set of data comparing the nanoparticle release of GO with varying filler weight concentration and correlating it the mechanical influence of the fillers. The results show that the release characteristics and the influence in particle number concentration are primarily dependent on the matrix brittleness and not necessarily the filler weight concentration within the nanocomposite. Keywords: nanoparticle ; release ; graphene ; oxide ; drilling ; epoxy ; nanocomposite ; carbon-fiber 1. Introduction Over the last couple of decades, epoxy-based (EP) nanocomposites have undergone intensive research and development ensued by their increasing implementation within commercial applications [ 1 ]. Carbon-fiber (CF) reinforced-EP composites have become well-established materials within various lightweight applications, most prominently the aeronautical and automotive industry. Nano-sized graphene oxide (GO) has been shown to improve and tailor the EP/CF matrix mechanical properties, as demonstrated in numerous studies [ 2 , 3 , 4 , 5 ]. However, the unique advantages and influence that GO nanoparticles have on the material properties accompany potential exposure to unique toxic effects within biological systems. Carbon-based nanofillers are of particular interest within the nanotoxicity literature due to the exceptional material properties and wide use across industries. Consequently, GO has demonstrated potential toxicological and/or eco-toxicological hazard [ 6 , 7 , 8 , 9 , 10 , 11 ]. Although the nanoparticles are initially embedded within the nanocomposites, a full understanding of the inadvertent release of nanoparticles within the workplace and atmosphere poses unknown potential hazards which are yet to be fully understood or quantified [ 12 ]. EP is one of the most utilized thermosets within polymers, with an estimated global EP resin market to be USD 10.6 billion by 2023 at a compound annual growth rate of 5.24% between 2017 and 2023 [ 13 ]. A separate report on global fiber reinforced composites forecasts a compound annual growth rate of 8.20% between 2018 and 2024 [ 14 ]. The use of carbon nanofillers to improve interfacial bonding between CFs and the polymer matrix is widely demonstrated in the literature with the use of GO [ 3 , 15 , 16 ]. Throughout its use, a polymer nanocomposite will undergo industrial machining where drilling, along with other machining scenarios, can lead to material damage and/or the release of potentially toxic nanoparticles [ 17 , 18 ]. Three excellent review studies on the release and/or exposure of nanoparticles due to lifecycle scenarios on nanocomposite materials have highlighted similar findings that high-quality evidence has demonstrated nanoparticle exposure, which is relevant during machining [ 18 , 19 , 20 ]. One study [ 19 ] concluded that whilst data currently indicate a high portion of the release to be partially or fully embedded nanomaterials, there is a shortage of research into the release of manufactured nanomaterials. In another study [ 18 ], the authors concluded that although there is a lack of data for engineered nanomaterial (ENM) exposure and with limitations between data, the literature suggests that all three routes of exposure (i.e., inhalation, dermal and ingestion) are relevant for workers in the manufacturing of ENMs. One potential route for exposure is during the composite and component manufacturing stages involving processes such as drilling for joining, integration and assembly of parts [ 21 ]. For example, approximately 180,000 holes are drilled to produce a single Airbus A380 wing, and around 60% of rejected parts are due to defects introduced in holes [ 22 ]. Composites drilling is therefore an important operation at the manufacturing stage that leads to the generation of dust and potential nanoparticle release. From the current literature, only nine studies [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ] have been identified to have investigated the nanoparticle release from drilling in nanocomposite materials. All nine of the studies on drilling demonstrated nanoparticles to be released. However, whilst some studies demonstrated a 56-fold increase in nanoparticle release due to the addition of SiO 2 [ 26 ], or a 211% increase with the addition of CNTs [ 29 ], some studies showed smaller influences, such as one study [ 27 ] which utilized a 0.09 wt.% SiO 2 in polyurethane and only observed a minimal increase in particle number concentration. Noticeably though, studies have not investigated the influence of varying weight concentration of the nanoparticle filler on nanoparticle release. Nonetheless, most studies concluded the nanoparticle fillers to have a statistically significant influence on the nanoparticle release, but observed significant differences between studies in the particle number concentrations. The findings from the nine studies on the influence of nano-sized fillers on nanoparticle release during drilling are in general agreement with the lack in knowledge and the need for a harmonized methodology in order to compare the materials and processes that are reported in the literature on nanoparticle release from machining. 14 ], the materials will undergo drilling during the assembly and manufacturing stages. As evident within several studies, composite materials with nanoparticles have shown potential release of the nanoparticles [ 18 , 19 , 20 ]. This study will therefore investigate the influence of the GO nanoparticles on the nanoparticle release during the identified release scenario, drilling. 2. Experiments 2.1. Materials Epoxy composites are vastly reported and used within industry to be reinforced with more conventional, micron-sized carbon fiber. The use of GO and other carbon-based nanofillers incorporated into hybrid CF and EP composites is less well known and embeds various nanofiller concentrations [ 32 , 33 , 34 ]. For this reason and based on studies within the literature [ 3 , 35 , 36 , 37 ], GO was chosen as a filler with concentrations of 0.05, 0.1 and 0.5 wt.%. A commercially available high-performance bisphenol-A-(epichlorhydrin)-based epoxy resin specifically formulated for use in vacuum resin infusion from Easycomposites, Stoke on Trent, UK (IN2 Epoxy Infusion Resin) combined with a polyoxypropylendiamin-based hardener from, Easycomposites, Stoke on Trent, UK (AT30 Epoxy Hardener –Slow) was chosen for the matrix. Graphene oxide (GO) flakes, 15–20 sheets with 4–10% edge-oxidized from Sigma-Aldrich, Dorset, UK (796034 Aldrich) were employed in this investigation. The 3k 2/2 twill woven carbon fiber was obtained from Easycomposites, Stoke on Trent, UK (Carbon Fibre 2/2 Twill 3k 210g). The composite samples were manufactured through the vacuum resin infusion method [ 5 ]. Concentrations of 0.05, 0.1 and 0.5 wt.% relative to the wt.% epoxy were initially dispersed within methanol with the use of a sonication bath for 1 hour to allow for later dispersion of the GO in the epoxy. Once fully dispersed, the solution was then homogenously dispersed within the bisphenol-A-(epichlorhydrin)-based epoxy and placed in a vacuum oven for 2 hours at 60 °C to allow for slow solvent evaporation. The solution was then mixed together with the hardener using a magnetic stirrer and manual mixing. This was followed by the vacuum resin infusion process with 6 layers of the carbon fiber textile layered within a mold and left to cure for 7 days at room temperature. A 60:40 fiber-epoxy volume ratio and epoxy resin to hardener (100:30) was employed as recommended by the supplier. A reference sample without any GO was also manufactured (EP/CF), along with 0.05 wt.% GO (EP/CF/GO 0.05 wt.%), with 0.1 wt.% GO (EP/CF/GO 0.1 wt.%) and with 0.5 wt.% GO (EP/CF/GO 0.5 wt.%) samples. 2.2. Automated Drilling Nanoparticle Release Methodology The methodology utilizes a process-related approach. This process is designed to simulate mechanical drilling on nanocomposite materials and is continued work from the Nanomaterials-Related Environmental Pollution and Health Hazards Throughout Their Life-Cycle (NEPHH) project study [ 25 , 26 ] funded by The European Commission (EC FP7 Project- CP-FP; Project Reference: 228536–2) and documented in three other studies [ 28 , 29 , 30 ]. A crucial factor identified in the literature and the Organisation for Economic Co-operation and Development (OECD) guidelines is for the methodology to make a distinction from the background or to control the background particles to setup a controlled environment. Building on the NEPHH project, the chamber is capable of achieving a clean environment monitored using a condensation particle counter (CPC), importantly removing all background noise or interference in the measurement of number concentration and particle size distribution. The data collected are therefore a representation of the particles released solely from the material. Removing the background data allows for a depiction of any particles released from the materials which can be directly linked as an unconditional maximum exposure assessment [ 38 ]. As proposed in several studies [ 39 , 40 , 41 ] with a controlled testing setup and environment, only one parameter, the material, is changed and investigated. This simplifies the issue of accounting for local background influences, as specified within the guidelines and reports by [ 42 , 43 ]. Once the chamber was cleared of any particles, the drilling studies were carried out by drilling across the width of the sample resulting in eight separate holes and bearing a time duration of 3 min of drilling, followed by 1 min of post-drilling. The eight holes drilled per sample were repeated three times to get an average of the particle number concentration and particle size distribution released. Based on previous studies carried out on nanocomposite drilling [ 24 , 25 , 26 ], a standard Dremel 4000 drilling tool with an industrial standard stainless steel 3.5 mm twist drill bit was used at 10,000 rpm with a feed rate of 78 mm/min. The setup uses an automated drilling assembly operated externally to the chamber to permit a repeatable and controlled environment within the chamber as shown on Figure 1 . The closed steel chamber has dimensions of 740 mm × 550 mm × 590 mm, and therefore a total inner volume of 0.240 m 3 . It is designed to assure a closed environment to simulate an appropriate volume around the drill and minimizing electrostatic attraction to the surfaces. To quantify only the particles released from the sample, the chamber was initially cleared of particles through an inflow of clean air with the use of TSI 99.97% retention HEPA Capsule Filters. A separate capsule was constructed around the drill with separate air flow to avoid any interference of the drilling fumes on the particle number concentration within the capsule. The clean air system using the HEPA capsule filters and with the drill on was capable of producing a particle number concentration reading within the chamber of 0 particles/cm 3 with false background counts < 0.01 particles/cm 3 , as measured using a TSI Environmental Condensation Particle Counter (CPC) model 3783 at a flow rate of 0.6 L/min, particle range of 7–3000 nm and concentration range of 0–10 6 particles/cm 3 and ± 10% at 10 6 particles/cm 3 . The level of background noise is therefore significantly within the ISO [ 44 ] cleanroom standard for particles ≥ 0.1 µm of 10 particles/cm 3 . An outlet channel is placed adjacent to the test specimen for the nanoparticle release equipment readings. A standard IOM Inhalable Sampler for the collection of inhalable particles was placed next to the test specimen with a 2 L/min suction to attract and prevent particles from detaching away from the grid for post-test chemical analysis [ 45 ]. An additional sampling tray was positioned below the test specimen for collection of the deposited particles for further post-test analysis. The scanning mobility particle sizer (SMPS) used for the study is a TSI 3080 Electrostatic Classifier utilizing a nano differential mobility analyzer (DMA) with 99 distinct particle diameters within a particle range of 4.61–156.8 nm and a flow rate of 0.31 L/min. The principle of the Model 3080 Electrostatic Classifier with the DMA is based on the monotonic relationship between electrical mobility and particle size with singly charged particles. The aerosol particles go through a process of bipolar charging or “neutralization” and are then classified with the differential mobility analyzer and then measured by a condensation particle counter. The given particle size distribution is therefore corresponding to the electrical mobility diameter. In addition, separate repeated runs were carried out using a Cambustion DMS50 Fast Particle Size Spectrometer with a 1 s sampling period, inlet flow rate of 6 L/min, with 34 distinct particle diameters of size range between 4.87 and 562.34 nm for the particle size distribution. The DMS50 utilizes a unipolar corona charger placing positive charges on each particle which are then classified along electrometer detectors based on mobility and hence particle size [ 46 ]. The charge is conducted via an electrometer amplifier whose output indicates the flux of particles giving the particle concentration at that given particle size. Since the classification of particles according to their differing electrical mobility takes place in parallel (rather than in series as in the SMPS), the DMS50 can offer the faster sampled particle size distribution. This allowed for a size distribution every second compared to the SMPS of 45 s period (followed by 10 s for the classifier to regenerate to its initial voltage and 5 s to start the size distribution again) and therefore a faster representation of the particles being released from the sample during drilling. The SMPS uses the assumption of spherical particles. Hence, from the diameters of the particle size distribution measured, and the material density of the nanocomposites, the particle mass size distribution can be estimated. Both the Zeiss EVO LS10 Variable Pressure Scanning Electron Microscope and an SEM/EDX (FEI Quanta 200F) with a beam current of 208 µA and voltage of 10 kV were used for the present study and cross-checked using an electron probe microanalyser (EPMA) JEOL JXA-8621MX, with a beam current of 30 nA and voltage of 15 kV. SEM samples of the materials were prepared using sputter coating of an ultra-thin coating of gold to minimize charging. A sampling tray placed immediately below the drilling set up in the chamber was used to collect debris removed from the nanocomposites during the drilling operation. 2.3. Mechanical Properties The influence of the addition of the GO nanofillers on mechanical properties (tensile and flexural) was evaluated and compared. To achieve this, the materials underwent a tensile test in accordance to ASTM D 3039/D tensile test [ 47 ] and 3-point flexural test in accordance with reference standard ASTM D 7264/M flexural test [ 48 ]. The tests were carried out with the use of an Instron 3382 universal testing system with a 100 kN load range. Raw data were collected using the Bluehill 3 software as measured in terms of load and extension. As per the respective standards, a constant head-speed of 2 mm/min for the tensile test and 1 mm/min for the flexural test was used, and data were collected at 10 Hertz. The data were then converted from the load and extension to stress vs. strain. 3. Results 3.1. Filler Effect on Mechanical Properties Following the tensile and flexural testing standards of polymer matrix composite materials, the respective tensile and flexural properties of the materials were determined. A comparison of the tensile and flexural properties is shown in Figure 2 . Statistical analysis was carried out on the samples. From a t-test between each sample and the EP/CF sample, only the EP/CF/GO 0.1 wt.% did not return a statistically significant difference for Young’s modulus (EP/CF/GO 0.05 wt.% p = 0.0000567, EP/CF/GO 0.1 wt.% p = 0.240, and EP/CF/GO 0.5 wt.% 2 wt.% p = 0.0391). The analysis on the tensile strength, returned all of the reinforced samples with a statistically insignificant difference (EP/CF/GO 0.05 wt.% p = 0.0814, EP/CF/GO 0.1 wt.% p = 0.222, and EP/CF/GO 0.5 wt.% 2 wt.% p = 0.175). This is attributed to the high deviation between samples. Therefore, whilst none of the samples returned a statistically significant difference in tensile strength, only the EP/CF/GO 0.1 wt.% did not return a statistically significant difference for Young’s modulus. As with the tensile results, statistical analysis was carried out on the flexural tests. From the t-test between each sample and the EP/CF, all of the samples returned a statistically significant difference in flexural modulus (EP/CF/GO 0.05 wt.% p = 0.00784, EP/CF/GO 0.1 wt.% p = 0.000622, and EP/CF/GO 0.5 wt.% 2 wt.% p = 0.0142). In the analysis of the flexural strength, only the EP/CF/GO 0.05 wt.% returned with a statistically insignificant difference (EP/CF/GO 0.05 wt.% p = 0.0575, EP/CF/GO 0.1 wt.% p = 0.0106, and EP/CF/GO 0.5 wt.% 2 wt.% p = 0.00765). Therefore, whilst the EP/CF/GO 0.05 wt.% returned with a statistically insignificant difference in flexural strength, the inclusion of GO returned statistically significant differences in flexural modulus and flexural strength, both negative and positive. The incorporation of the GO nanofiller had contrasting influence on the material properties. The influence of GO on mechanical properties will be correlated to the nanoparticle release. 3.2. Filler Effect on Particle Number Concentration The GO-reinforced EP/CF samples underwent the repeated drilling, and the particle number concentration was measured during the testing. Using the CPC, the particle number concentration was quantified in situ with a sampling rate of 1 s. An average of the repeated test ( n = 3) for each sample is displayed in Figure 3 . The eight holes drilled are clearly evident within the particle number concentration over the 4 min of data sampling. Eight peaks represent the eight holes drilled, followed by one minute on no drilling and the concentration stabilizes. A maximum value in terms of quantity of the particles being released at the time of drilling is obtained. The methodology is able to provide a comprehensive depiction of the particles released during the drilling before (anything within 1 s) dispersion and scattering within the chamber. The particle number concentration can then be observed to relatively stabilize during the 1 min after the drilling has ended but does not drop back to the initial 0 #/cm 3 . Thus, the particles produced from the drilling remain airborne within the chamber environment. These two meaningful annotations therefore epitomize the release characteristics observed: peak particle number concentrations and remaining airborne concentration after 4 min of sampling. The mechanical drilling therefore generates a substantial quantity of nanoparticles, which then quickly disperse, but remain airborne within the chamber (evident through stable concentration). From the comparison between the EP/CF sample and the reinforced samples with GO, any disparity between the samples is not clearly apparent. The peaks concentrations of release during drilling are spread across two peaks which can be associated to the drill bit entering and the withdrawal of the drill bit from the material. The withdrawal of the drill bit can clearly be evident of producing the higher particle number concentration. Within the averages, only the first hole of the EP/CF samples displayed a higher particle number concentration from the drill bit entering the material than during the withdrawal. However, the introduction of GO into the samples at the three different weight concentrations did not demonstrate any noticeable difference to the profile of the release during entering or withdrawing the drill bit. The data demonstrate that even the samples without the reinforcement of GO nanoparticles, released a substantial particle number concentrations during drilling. The EP/CF sample without any nanoparticles, observed a peak particle number concentration comparable to the samples reinforced with the GO nanoparticles. However, from the average profiles, the EP/CF/GO 0.5 wt.% sample can be seen to have obtained the highest peaks. These also can be seen to slowly increase in size over the eight holes, with the exception of the seventh hole. This would suggest the more holes being drilled also increases the particle number concentration peak size. Therefore, GO at 0.5 wt.% shows an increasing trend with more holes drilled, which is not evident within other studies [ 28 , 29 , 30 ]. Whilst the 0.5 wt.% GO is understood to increase the peak particle number concentrations released during drilling (with a 243% increase in mean peak particle number concentration), the two other GO weight concentrations have contrasting effects. The peaks introduced from the EP/CF/GO 0.1 wt.% followed a comparable profile to the peaks from the EP/CF samples, whereas the EP/CF/GO 0.05 wt.% released some slightly higher peak concentrations. A numerical and statistical representation of the data from all samples is shown in Table 1 . Table 1 displays the statistical analysis carried out on the peak particle number concentrations of the samples. The calculated lower tail of 5% and upper tail of 95% give a representation of the data for a 90% confidence interval of a t-distribution. This highlights the disparities between the peak particle number concentrations and therefore, a statistically significant difference with the introduction of GO on release in comparison to the EP/CF. A two-sample t-test of significance of each sample mean and deviation to the neat EP/CF sample returned statistically significant differences for all concentrations of GO (outside the 90% confidence interval). ANOVA single-factor analysis was performed to assess the variability between the sample peak means introduced due to the fillers. The analysis returned statistically significant differences within the four samples (F value = 4.63 F critical value = 2.95) and a 0.946% chance that the observation could have been observed due to random error alone, therefore rejecting a hypothesis that the samples displayed no difference. It is important to note that although the statistical analysis returned a statistically significant difference with the introduction of the GO, this does not embrace the extent of the difference. From the data represented in both Table 1 and Figure 3 , the incorporation shows a minor influence on the increase in particle number concentration. With the comparison of the samples, the EP/CF/GO 0.5 wt.% demonstrated a clear increase in all aspects of the particle number concentration, whereas the 0.05 and 0.1 wt.% displayed a more minor increase in peak particle number concentration values. As with all other samples, the statistical analysis does consider the high standard deviation and range demonstrated and therefore includes the level of randomness and variability in the peaks released. From the numerical values, the EP/CF/GO 0.5 wt.% reinforced sample exhibited the uppermost mean value over the 4 min with 1.07 × 10 4 #/cm 3 introduced into the chamber during drilling. In relation to the EP/CF, the EP/CF/GO 0.05 wt.% and EP/CF/GO 0.1 wt.% produced a difference in nanoparticle concentration of 31.9% and −1.17% on average over the 4 min, respectively. Therefore, although the EP/CF/GO 0.05 wt.% and EP/CF/GO 0.5 wt.% observed an increase in particle number concentration over the 4 min, the EP/CF/GO 0.1 wt.% demonstrated a slight decrease. Furthermore, to correlate the increasing weight concentration of GO on nanoparticle release, no statistical model can be created. This is due to an increase in concentration with 0.5 wt.% GO followed by a decrease from the 0.1 wt.% GO, and finally a larger increase from the 0.5 wt.% sample. The correlation therefore does not follow a trend or correlation between weight concentration and particle number concentration released. It is notable that while the EP/CF/GO 0.5 wt.% sample produced the highest concentration and peaks during the drilling, it also demonstrated the highest concentration at the end of the four-minute sampling period. The high particle concentration introduced during the drilling indicates that the particles disperse within the chamber but crucially remain airborne. The EP/CF/GO 0.5 wt.% sample presented a particle number concentration remaining above 1.2 × 10 3 #/cm 3 even after the drilling and 1-min post drilling was concluded. The graphical representation of the average particle number concentration measured at the end of the four-minute sampling period is presented in Figure 4 . The particle number concentration measured at the end of the sampling period is beneficial to evaluate the effect of the filler on the rapidity of depositing and dispersion within the chamber. The difference in particle concentration at the time of release due to the holes and concentration at the end of the sampling period presents an indication into these properties. The EP/CF/GO 0.5 wt.% sample observed a 118% increase measured at the 4th min from the EP/CF sample in comparison to the 112% increase over the previous four minutes (EP/CF/GO 0.05 wt.% increase of 44% and EP/CF/GO 0.1 wt.% increase of 5% from the EP/CF sample measured at the 4th minute). The difference at the 4th minute being similar to that measured over the four minutes demonstrates that the deposition rate during the 1-min post drilling is similar between all samples. Therefore, as well as demonstrating the highest peak particle number concertation released during drilling, the particles released from the EP/CF/GO 0.5 wt.% do not deposit any quicker and remain airborne to observe the highest particle number concentration post drilling. 3.3. Filler Effect on Particle Size Distribution Simultaneously to the data gathered for the particle number concentration, the particle size distribution was quantified in situ using the SMPS and the DMS50. This provides a better understanding of the size of the particle number concentration seen in the Figure 3 . An average of the four 1-min sampling periods measured across the four minutes is represented in Figure 5 . From the distribution, the EP/CF/GO 0.5 wt.% can be seen to have the most substantial effect on the particle size distribution. Two large peaks are observed on the limits of the SMPS between particle diameters of 4 to 6 nm and 80 to 100 nm. All the other samples observed smaller peaks, and at different particle diameters. The size distribution illustrates minimal effect with the introduction of GO nanofillers at 0.05 and 0.1 wt.% in comparison to the EP/CF sample. Excluding the EP/CF/GO 0.5 wt.%, the size distribution shows a relative scatter across the 100 nm spectrum. Slight increases are observed at 18 nm and between 40 and 50 nm, but these are still unparalleled compared to the peaks observed from the EP/CF/GO 0.5 wt.% sample. The peaks observed below 6 nm from the EP/CF/GO 0.5 wt.% are quite significant in magnitude and substance. The GO embedded within the EP/CF consists of 15 to 20 sheet flakes which will therefore have a thickness of up to 20 nm. Each GO sheet can have a thickness of around 1 nm (796034 Sigma Aldrich, Dorset, UK). Drilling creates shear forces within the material which can therefore be related to possible separation of the layers due to the drilling. Furthermore, the EP/CF sample without any nanofiller did not exhibit any release peaks at these diameters. It is possible therefore, but only as a presumption, that the peaks observed below 6 nm could be associated to the GO fillers. However, this cannot be confirmed without identification of the independent GO fillers and peaks at the original thicknesses of the GO would be expected at around 20 nm. The peak observed around 100 nm does not correlate with either of the individual fillers. The CF fibers have a thickness within the micron-range and were not apparent in the particle size distribution of the EP/CF sample. Any independent CF or matrix-filler (EP and CF) embedding released from the samples would be expected within the EP/CF sample. The peak may be associated to either agglomerations of the GO nanoparticles or GO embedded within the matrix. However, both would also be expected within the other GO reinforced samples, unless the higher weight concentration is likely to increase the separation of the GO from the CF. Nonetheless, the EP/CF/GO 0.5 wt.% is deduced to have influenced the particle size distribution quite significantly. In comparison, however, the EP/CF/GO 0.05 wt.% and EP/CF/Go 0.1 wt.% observed minimal influence on the particle size distribution in contrast to the EP/CF sample. Further to the data collected on the SMPS, separate data were gathered on the DMS50 for the size distribution at each second and is displayed in a 3D plot as demonstrated for the 0.1 WT.% sample in Figure 6 (Note: data were taken from a separate run to the CPC and SMPS data due to the required increased inflow rate). As demonstrated in the CPC data previously shown in Figure 3 , the DMS50 data display the peaks introduced during drilling across the first three minutes, followed by the post-drilling minute with reduced particle number concentrations. The eight peaks represent the eight holes drilled. This is apparent in all of the samples reinforced with GO, although also slightly less obvious. Due to the relatively low particle number concentrations, the peaks during drilling are less apparent. Similarly, the particle size distribution is not clearly evident in the one-minute post-drilling and the concentrations between drilling. The difference between the samples is similar to the CPC data, where the EP/CF/GO 0.5 wt.% sample exhibited the highest particle number concentration, which is conveyed into the DMS50 data. Lower peak concentrations are observed for the other samples, with relatively lower concentrations after drilling. As a result, the DMS50 data concur with the CPC data on the influence of the GO on particle number concentration. It is noticeable within all samples, and as demonstrated in the CPC data, that the peak particle number concentrations introduced during the drilling are relatively inconsistent, followed by a more stable and consistent post-drilling concentration. Although the particle size distributions introduced during the peaks from drilling are different between samples, the distributions are relatively consistent within each sample. The particle size distributions can therefore be associated to the material, as opposed to any factor related to the continuation of the drilling, such as the particles present or the number of holes already been drilled by the drill bit. A two-dimensional plot of the average particle size distribution introduced at the peaks due to drilling will therefore be representative of the eight holes drilled for each sample and is presented in Figure 7 . The four samples displayed contrasting results in particle size distribution released at the moment of drilling. Although demonstrating different peak sizes, a relatively high proportion of the size distribution from the four samples is ascertained to be between 40 and 100 nm in particle diameter. Whilst the EP/CF sample did not display a discrete sharp peak, all other samples revealed the highest peak within this same size range. This is, however, the one similar element visible in the four samples. The sample without any GO nanoparticles observed a broad range of particle diameters. Similarly, the peaks exhibited from the EP/CF/GO 0.05 wt.% were split across a 10 to 70 nm particle diameter. In contrast, the EP/CF/GO 0.5 wt.% displayed two peaks, one between 40 and 100 nm and one between 150 and 400 nm in particle diameter. In comparison to the SMPS data of the particle size distribution, the broad range and variation in particle diameter for the EP/CF, EP/CF/GO 0.05 wt.% and EP/CF/GO 0.1 wt.% samples are in moderate agreement with the DMS50 data. The peak observed at around 100 nm from EP/CF/GO 0.5 wt.% sample is somewhat similar to the SMPS data, however, the DMS50 did not display a peak for the sample at lower concentrations. Similar to the data presented in previous chapters, the disparate peaks seen on the two instruments introduce debatable deductions and limited effectiveness of instrumentations required for real-time data. Nonetheless, the data from the DMS50 demonstrated no evidence of independent nano-sized GO fillers within any of the particle size distributions for the GO reinforced samples. With almost no peaks apparent with a particle diameter less than 10 nm, the suggestion of GO layers separation due to the drilling shear forces is not evident in the DMS50 data. However, the GO is shown to increase the particle number concentration between 40 and 100 nm. The source of the increase is the higher particle number concentration observed in the GO reinforced samples. However, due to the particle size diameters, these cannot be associated to independent nanofillers, and instead either agglomerations or matrix-filler embedded particles. Nonetheless, all three instruments used to quantify the released particles (CPC, SMPS and DMS50) demonstrate a harmonized maximum increase in particle number concentration from the EP/CF/GO 0.5 wt.% sample. 3.4. The Filler Effect on Mass Size Distribution Since the drilling was conducted without any interference from background particles, all of the particles measured on the instrumentation are from the nanocomposite material. With the use of the SMPS and the known density of the individual nanocomposites, the particle mass concentration can therefore be estimated. The data utilize the diameter of the particles measured using the SMPS. The constant material density for the three nanocomposites are: EP/CF = 1.59 g/cm 3 , EP/CF/GO 0.05 wt.% = 1.59 g/cm 3 , EP/CF/GO 0.05 wt.% = 1.59 g/cm 3 and EP/CF/GO 0.5 wt.% = 1.57 g/cm 3 . The average mass concentration across the 4-min sampling period for different particle size diameters is illustrated in Figure 8 . The particle diameters with high particle number concentrations observed in the SMPS results have been adjusted due to the consequent mass increase of larger particles. Almost no significant peak was perceived below 50 nm. All of the samples consequently displayed an increase in particle mass concentration in diameters larger than 50 nm up until the SMPS limit of approximately 157 nm. As with the particle size distribution, the EP/CF/GO 0.5 wt.% demonstrated the largest peak at around 100 nm. The remaining samples recorded similar relative peaks between 50 and 157 nm. The EP/CF/GO 0.05 wt.% and EP/CF sample displayed a similar increasing profile in particle mass concentration over 100 nm. As with the particle number concentration and particle size distribution, the EP/CF/GO 0.5 wt.% clearly demonstrated an augmenting effect in concentration, with similar mass concentrations for the remaining EP/CF, EP/CF/GO 0.05 wt.% and EP/CF/GO 0.1 wt.% samples. Since the CPC can measure a larger particle size range, an alternative mass concentration is valuable to quantify the release. Using the particle number concentration measurement at the end of the four-minute sampling period, and the calculated total quantity of mass drilled, an estimation of the concentration of particles/mass drilled can be acquired and is presented in Figure 9 . This is calculated using the particle number concentration of the CPC (size range: 7 to 3000 nm), material density values and equivalent of mass drilled based on hole size and number of holes. The number of particles to mass drilled ratio also presents the EP/CF/GO 0.5 wt.% sample with the highest particle release over the EP/CF and other GO reinforced samples (EP/CF = 3974 #/cm 3 g drilled , EP/CF/GO 0.05 wt.% = 5702 #/cm 3 g drilled , EP/CF/GO 0.1 wt.% = 4167 #/cm 3 g drilled , and EP/CF/GO 0.5 wt.% = 8758 #/cm 3 g drilled ). Since the density of the EP/CF/GO 0.05 wt.% and EP/CF/GO 0.1 wt.% sample did not change sufficiently to be measured with the addition of the GO, the correlation to the EP/CF sample is the same as the particle number concentration previously presented. However, the slight decrease in density in the EP/CF/GO 0.5 wt.% sample means that the sample observed a 118% increase in normalized total concentration over the EP/CF sample. The particle mass concentration is an important identified parameter within the literature when evaluating the release or exposure to nanoparticles. The data identify important differences and support the findings on the effect of the filler on particle number concentration and particle size distributions. The GO at lower weight concentrations shows minimal effect on the release, whereas the EP/CF/GO 0.5 wt.% sample displayed a significant difference in comparison to the EP/CF sample. 3.5. Assessment of Deposited Particles The debris collected in the chamber as described in the methodology was analyzed using an SEM. An SEM image of the debris for each sample is displayed in Figure 10 . A larger magnification of the dust collected in the sampling placed underneath the drilling is shown in Figure 11 . The deposited particles collected illustrate a large variation, such as particles, agglomerates and independent fibers and matrix. The image has a relatively distant magnification, which allows us to display the micro-sized CFs and particle aggregation at a micro level. The nanoparticles are therefore not distinguishable and are shown in Figure 11 . Within the microscopy analysis of all the GO reinforced samples, no independent GO nanoparticles were identified. The GO reinforced samples were instead seen to demonstrate an increase in particles embedded or adhered to the surface of the CFs, as can be seen in Figure 11 . The neat EP/CF sample displayed significantly fewer particles attached onto the independent fibers identified within the deposited particles. The few particles attached onto the CF shown in Figure 11 a can be attributed to the EP, as no GO has been added. The GO-reinforced samples, however, demonstrated visibly more particles on the surface of the CFs. This can be attributed to either the GO particles or the EP. As is observed in the literature, GO particles are seen to improve the interfacial bonding between the CF and the EP. The microscopy images of the surface of the carbon fibers with attached particles of GO and EP are in accordance with similar findings in other recent studies that embedded GO within EP/CF samples [ 49 , 50 ]. The increase in the number of particles attached to the deposited fibers was the only identifiable difference in the morphology studies investigating the influence of the GO filler weight concentration. The deposited particles therefore observed no identifiable independent GO nanoparticles, and instead were seen to increase the particles bonding to the surface of the CFs. The findings within deposited particles, therefore, do not aid in identifying the source of the noted increase in the particle size distribution and particle mass distributions at 100 nm. Further, the deposited particles do not provide evidence of independent GO nanoparticles released from the embedding within the nanocomposite materials. The data, however, are a representation of the deposited particles collected within the sampling tray, and not the measured airborne particles through the particle quantification instruments. Within the deposited particles, the release is shown to be matrix or CF-orientated with GO embedded or adhered to the surface. 4. Discussion 4.1. Influence of Filler From the three samples investigated with the incorporation of nanofillers, all three demonstrated a statistically significant difference in a two-sample t-test of significance of each sample mean and deviation to the reference sample (test of 90% confidence interval). It is important to note that although the statistical analysis returned a statistically significant difference with the introduction of the GO, this does not include the extent of the difference. The incorporation indicates a minor influence in the increase in particle number concentration. With the comparison of the samples, the EP/CF/GO 0.5 wt.% demonstrated a clear increase in all aspects of the particle number concentration; whereas the 0.05 and 0.1 wt.% displayed a more minor increase in peak particle number concentration values. As with all other samples, the statistical analysis does consider the high standard deviation and range demonstrated and therefore includes the level of randomness and variability in the peaks released. However, the different weight concentrations of GO within EP/CF samples revealed that there is no direct correlation between weight concentration within the nanocomposite and influence on particle number concentration. The samples displayed an increase in particle number concentration with 0.5 and 0.05 wt.% GO, but a minimal effect with 0.1 wt.% GO (EP/CF/GO 0.05 wt.% = 43% increase, EP/CF/GO 0.1 wt.% = 5% increase, and EP/CF/GO 0.5 wt.% = 118% increase). In view of the fact that the GO reinforced samples demonstrating the filler weight concentration within the nanocomposite alone does not correlate to the influence particle number concentration (i.e., increase in weight concentration does not demonstrate an equivalent increase in particle number concentration), the influence on particle number concentration can be compared with the influence on mechanical properties. With all other parameters unchanged, the only change in parameter is the nanocomposite composition. The comparison between the influence of reinforcement fillers with the EP/CF without GO reinforcement is shown in Figure 12 . The use of GO as a nanofiller displayed conflicting effects on the mechanical properties and particle number concentration from the EP/CF hybrid composites. The EP/CF/GO 0.05 wt.% sample displayed the most significant improvement in mechanical properties with a statistically significant increase in flexural strength, flexural modulus and Young’s modulus in comparison to the EP/CF sample. This resulted in a statistically significant increase in particle number concentration, but lower than the increase introduced from the EP/CF/GO 0.5 wt.% sample. The 0.1 wt.% GO observed a decrease in all mechanical properties, whilst the 0.5 wt.% GO displayed a statistically significant increase in both Young’s and flexural moduli but a decrease in related strengths. These results contrast with several studies that showed an increase in mechanical properties with low weight concentrations of GO and a decrease after a threshold quantity. Another study [ 51 ] found the peak mechanical performance at 0.3 wt.% GO with a clear decrement in properties from 0.4 wt.% GO. In contrast, a different study [ 15 ] observed maximum flexural properties at 0.5 wt.% GO, followed by a decline. However, no studies were found to have reported on the mechanical properties with concentrations lower than 0.1 wt.% GO added to EP/CF and this is therefore the first reporting of such. Similarly, no studies have reported on the release of GO from EP/CF hybrid composites and this is therefore also the first reporting of such to the authors best knowledge. Among the literature, the improvement to the bonding between the CF and polymers has shown that oxidative treatments or particles that can generate –OH or –COOH groups on the fiber surface will act as coupling or bonding agents [ 52 ]. GO can effectively enhance the interfacial adhesion as the sp2 structure of the GO is prone to attach onto the surface of the CF by π–π stacking interaction [ 53 ]. The improved bonding and interfacial adhesion between the matrix, GO and CF allows for an optimization in stress transfer between the softer matrix of the polymer phase, to the CF [ 2 ]. The limit of GO content is said to be at the point where GO initiates to bond with the hardener and hence prevent the interface between the epoxy and hardener. The cross-linking therefore is reduced, resulting in a weaker interfacial interaction [ 51 ]. The peak concentration was not evident in either the material properties or nanoparticle release. The 0.1 wt.% GO sample demonstrated the least influence on mechanical properties and also nanoparticle release. However, improvement in mechanical properties observed from the 0.05 and 0.5 wt.% GO samples did not show a correlation to the particle number concentration increase. Therefore, although the GO nanoparticles can be seen to influence the particle number concentration and mechanical properties, no correlation between weight concentration and the subsequent mechanical properties or nanoparticle release are evident. As discussed, currently only nine studies have evaluated the influence of nanoparticles on nanoparticle release during drilling [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. All of the studies observed nanoparticle release and highlighted the potential hazard and exposure to humans which needs to be understood. As with the conclusions within the literature on the influence of nanoparticles on nanocomposite material properties [ 54 ], the correlation between nanoparticles and effect on nanoparticle release is challenging. The results are in unison with multiple reports in the literature, as eluded to earlier on, in the complexity of nanoparticle release data from the nanoparticle fillers. A noteworthy observation by Hankin and Read [ 55 ], appropriate to the findings within this study, however relating to the current knowledge of risks associated with nanotechnology, noted that “research conducted to date has shown the potential risks of nanotechnologies to be associated with a high degree of complexity and uncertainty, with no clear-cut cause-and-effect relationship”. Although there is currently no predictive model of the release of nanoparticles from nanocomposite during drilling, the closest literature is on the production of emissions from drilling in metals [ 56 , 57 , 58 ]. The studies reported the fracture of the material to be highly associated to the brittleness of the material. The GO nanoparticles used within this study had minimal influence on the brittleness and ductility of the materials in comparison to the reference EP/CF without the nanoparticle fillers. Although the nanoparticles were observed to have an influence on some of the material properties, overall no significant influence on the brittleness of the material was observed, which might explain the lack of clear correlation observed in nanoparticle release with increasing weight concentration of GO. 4.2. Influence of Matrix As the same methodology was used in three other studies [ 28 , 29 , 30 ], the materials used across the studies can be compared. In contrast to EP-based samples from one study [ 29 ] without CF reinforcement, the peak particle number concentrations in this study can be seen to be significantly lower. Furthermore, a comparison with the materials utilized in other studies [ 23 , 24 , 25 , 26 , 27 , 31 ] highlights a significant contrast in particle number concentrations observed across the studies. Ranging from a peak concentration of around 7.5 × 10 3 #/cm 3 [ 28 ] compared to peaks of over 2.2 × 10 9 #/cm 3 [ 27 ], this demonstrates how the polymer might be a major factor in the particle number concentration, with differences of up to 10 6 #/cm 3 . However, this should also be taken with caution, as studies have used different drilling setups, parameters, lab environments and measurement techniques. Nonetheless, evaluating the general material brittleness and ductility (i.e., correlation to point of failure and plasticity observed) from the mechanical properties associated to the groups of materials used, a closer correlation to the nanoparticle release can be acknowledged. The interaction between the drill and material at the microstructure level effect the form of the chips created. Therefore, the material fracture mechanics and plasticity deformation could help to predict how the material will behave at the local stress levels induced by the drilling and can thus be associated with the characteristics of nanoparticle release. In comparison to another study [ 28 ], a thermoplastic and more ductile properties (polypropylene) observed a much lower particle number concentration than the brittle thermosets of EP/CF samples observed within this study. The high energy release when subject to stress of the brittle materials causes a considerably higher particle number concentration. The material ductility therefore indicates an influence on the nanoparticle release. This is similar to the conclusions in the studies on metallic material drilling [ 56 , 57 , 58 ]. Although the introduction of the nanoparticles at the varied weight concentrations within this study is shown to have an effect on the particle release in comparison to the EP/CF, the basic profile of the release did not observe a significant change (unlike a comparison of the data with other studies). Therefore, whilst the nanoparticles might have an effect on the nanoparticle release, the substantial profile of the release is indicated to be dictated by the polymer and larger filler weight concentrations. This could be compared to the influence the nanoparticles have on the mechanical properties. Whereas the nanofillers were shown to have an influence on the mechanical properties (i.e., up to 65%), most of the mechanical properties are inherited from the EP/CF without the nanoparticle reinforcement. Due to the low weight concentrations, the mechanical properties are highly driven by the matrix, which can be seen in the nanoparticle release. If the release is matrix oriented, this might have an effect on the health concerns arising from the use of the nanoparticles. Although some studies have demonstrated no increased toxicity [ 59 , 60 , 61 ], there is still a lack of understanding whether most embedded nanoparticles within the matrix are toxic, as they have not been investigated due to the complexity and variations in material phases [ 19 , 20 ]. The toxicity studies previously mentioned within this study report the understanding and toxicity of only the individual GO nanoparticles as opposed to a matrix/filler combination. Additionally, the identification of release of the embedded hazardous nanoparticles must also be linked to the exposure of the released particles for toxicological assessments [ 38 ]. As with particles exposed to human cells through inhalation, and ingestion, the literature has reported it to be necessary to study each nanoparticle individually to fully understand the toxicity effects [ 62 , 63 ]. The particle size has been highlighted to be one of the most influential material properties affecting toxicity, but is one of only many physio-chemical properties, such as shape, aspect ratio, surface area, chemical composition and crystalline structure, of nanoparticles that have been identified to influence the nanoparticle toxicity [ 63 ]. Additional to the concentration or potential dosage, this study reports the particle size, mass and morphology, which can all contribute to the toxicity [ 43 ]. As the matrix can be associated to have a significant effect on the particle number concentration, the particle size distributions observed similar dependency on the matrix. The use of the GO fillers introduced minor shifts in peaks with the introduction of the diameters. Furthermore, the data presented within this study are a representation of particle release from a process-related approach [ 38 ]. The methodology used a clean environment through the removal of all background interference. The data collected are therefore a representation of the particle emissions solely from the material. Removing the background data allows for a depiction of any particles released from the materials which can be directly linked as an unconditional maximum exposure assessment [ 38 ]. The results represent a worst-case scenario of potential nanoparticle atmospheric emissions from the materials. The removal of any background particles provides a clean environment to be able to evaluate the full release from the investigated materials. Particle background interference will differ in all lab environments and could influence/affect the particles release. The data provided allow for a comparison and evaluation of the material with and without the nanoparticle fillers and can be used to identify whether release is likely. The full extent of exposure or intensity in a workplace scenario or influence with atmospheric air could potentially differ and should therefore be evaluated. The results therefore represent the potential release of the fillers and do not represent the exposure. As discussed, in the literature [ 18 ], the identification of potential release is necessary in relation to the materials and given scenario. Other exposure determinants that may be important, such as personal behavior, experience, maintenance of hoods/ventilations, as well as housekeeping practices, will need to be considered when using the data to determine any exposure controls. 5. Conclusions The aim of this study is to investigate the influence GO has on nanoparticle emissions from EP/CF hybrid ENMs during drilling. Four EP/CF based composites were manufactured with three variations in weight concentrations of GO; 0.05, 0.1 and 0.5 wt.%. The influence of the GO nanoparticle weight concentrations has on nanoparticle release during drilling was investigated and correlated to the influence on mechanical properties. All samples demonstrated nanoparticle release, including the neat EP/CF sample without any GO nanoparticles. The results from the study offer a novel set of data comparing the nanoparticle release of GO with varying nanoparticle filler weight concentration and correlating it with the mechanical influence of the fillers. From the results, different conclusions were deduced. All three nanoparticle reinforced samples demonstrated a statistically significant difference in comparison to the EP/CF sample. The GO-reinforced samples demonstrated that the filler weight concentration within the nanocomposite alone does not correlate to the influence particle number concentration (i.e., increase in weight concentration does not demonstrate an equivalent increase in particle number concentration). Although a two-sample t-test of significance of each sample mean and deviation to the neat EP/CF sample returned statistically significant differences for all concentrations of GO (outside the 90% confidence interval), the inclusion of 0.05 and 0.1 wt.% GO nanoparticles demonstrated minimal effect on nanoparticle release. However, the EP/CF/GO 0.5 wt.% demonstrated a 243% increase in mean peak particle number concentration introduced during drilling. Similarly, at the end of the four-minute sampling period, the EP/CF/GO 0.5 wt.% sample observed a 118% increase in comparison to the EP/CF sample. However, the minor increases observed for the lower weight concentrations of GO reinforced samples do not substantiate an increase in particle number concentration with an increase in GO nanoparticles (EP/CF/GO 0.05 wt.% increase of 44% and EP/CF/GO 0.1 wt.% increase of 5% from the EP/CF sample measured at the 4th minute). Nonetheless, the statistical analysis returned a statistically significant difference with the introduction of GO nanoparticles within the nanocomposites on nanoparticle release during drilling. The particle size distribution illustrated minimal effect with the introduction of GO nanofillers in comparison to the EP/CF sample. Due to the particle size diameters, the peaks cannot be associated to independent GO nanofillers, and instead either agglomerations or matrix-filler embedded particles. Correspondingly, the assessment of the deposited particles displayed no evidence of independent GO nanoparticles. The comparison between the nanoparticle release and mechanical properties demonstrated no observable correlation with the samples used within this study. However, the comparison of the results with other studies highlights the potential for a correlation of the nanoparticle release and the matrix material properties. More precisely, the comparison between matrices highlighted that the majority of the release characteristics were indicated to be dependent on the material polymer brittleness. A comparison of the data leads towards a conclusion that the more brittle the material is, the higher the particle number concentration. The relatively minor influence that the GO nanoparticles have on mechanical properties exhibit similar minor, yet still statistically significant, influences on the nanoparticle release. Nonetheless, although the GO nanofillers are concluded to have demonstrated an influence on the nanoparticle release during drilling, the materials demonstrated a level of complexity with no clear cause and effect relationship. However, the possible dependence on polymer properties and correlation between the nanoparticle release and matrix mechanical properties may be used when improving materials and has the potential to act as part of developing nanocomposite materials as a concept of safety by design. Author Contributions Conceptualization, K.S. and J.N.; methodology, K.S. and J.N.; software, K.S. and J.N.; validation, K.S. and J.N.; formal analysis, K.S. and J.N.; investigation, K.S. and J.N.; resources, K.S. and J.N.; data curation, K.S. and J.N.; writing—original draft preparation, K.S. and J.N.; writing—review and editing, K.S. and J.N.; visualization, K.S. and J.N.; supervision, J.N.; project administration, J.N.; funding acquisition, J.N. All authors have read and agreed to the published version of the manuscript. Funding The work is funded by and part of the European Commission Life project named Simulation of the release of nanomaterials from consumer products for environmental exposure assessment (SIRENA, Pr. No. LIFE 11 ENV/ES/596). We are also thankful to the funding by QualityNano Project through Transnational Access (TA Application VITO-TAF-382 and VITO-TAF-500) under the European Commission, Grant Agreement No: INFRA-2010-262163 for the access and use of the facilities at the Flemish Institute for Technological Research (VITO). Acknowledgments The authors are grateful to the funding bodies and would also like to acknowledge E Frijns, J Van Laer, K. Tirez and R. Persoons at VITO NV (Mol, Belgium) for their support during nanoparticles release measurements, XRF and SEM characterisation. Conflicts of Interest The authors declare no conflict of interest. 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Design drawing of drilling setup within enclosed test chamber with cycled airflow to allow for a clean environment removing any background interference represented (not to scale). Figure 2. Comparison of mean values of epoxy-reinforced carbon fiber (EP/CF)-based samples reinforced with graphene oxide (GO) of ( a ) tensile properties and ( b ) flexural properties. Figure 2. Comparison of mean values of epoxy-reinforced carbon fiber (EP/CF)-based samples reinforced with graphene oxide (GO) of ( a ) tensile properties and ( b ) flexural properties. Figure 3. Particle number concentration averages of nanoparticles introduced from EP/CF-based samples reinforced with GO and measured using condensation particle counter (CPC) ( n = 3 for each average). Figure 3. Particle number concentration averages of nanoparticles introduced from EP/CF-based samples reinforced with GO and measured using condensation particle counter (CPC) ( n = 3 for each average). Figure 4. Particle number concentration recorded at 4th min (#/cm 3 ) for EP/CF-based samples reinforced with GO as measured on the CPC ( n = 3 for each average). Figure 4. Particle number concentration recorded at 4th min (#/cm 3 ) for EP/CF-based samples reinforced with GO as measured on the CPC ( n = 3 for each average). Figure 5. Average particle size distribution measured using scanning mobility particle sizer (SMPS) of EP/CF-based nanocomposites reinforced with GO ( n = 12 for each average). Figure 5. Average particle size distribution measured using scanning mobility particle sizer (SMPS) of EP/CF-based nanocomposites reinforced with GO ( n = 12 for each average). Figure 6. Particle size distribution recorded on the DMS50 during 4 min from the EP/CF/GO 0.1 wt.% sample. Figure 7. Peak particle size distribution within the 4 min sampling of the EP/CF-based samples reinforced with GO recorded on DMS50. Figure 7. Peak particle size distribution within the 4 min sampling of the EP/CF-based samples reinforced with GO recorded on DMS50. Figure 8. Particle mass concentration average over 4 min of EP/CF based nanocomposites reinforced with GO determined from SMPS ( n = 12 for each average). Figure 8. Particle mass concentration average over 4 min of EP/CF based nanocomposites reinforced with GO determined from SMPS ( n = 12 for each average). Figure 9. Normalized total concentration of particles (C divided by estimated drilled mass) recorded at 4th min for EP/CF based samples reinforced with GO ( n = 3 for each average). Figure 9. Normalized total concentration of particles (C divided by estimated drilled mass) recorded at 4th min for EP/CF based samples reinforced with GO ( n = 3 for each average). Figure 10. Deposited particles collected in a sampling tray placed directly below drilling from the EP/CF/GO 0.5 wt.% sample. Figure 10. Deposited particles collected in a sampling tray placed directly below drilling from the EP/CF/GO 0.5 wt.% sample. Figure 11. SEM images of deposited particles collected in sampling tray from drilling on ( a ) EP/CF sample, ( b ) EP/CF/GO 0.05 wt.% sample, ( c ) EP/CF/GO 0.1 wt.% sample and ( d ) EP/CF/GO 0.5 wt.% sample. Figure 11. SEM images of deposited particles collected in sampling tray from drilling on ( a ) EP/CF sample, ( b ) EP/CF/GO 0.05 wt.% sample, ( c ) EP/CF/GO 0.1 wt.% sample and ( d ) EP/CF/GO 0.5 wt.% sample. Figure 12. Comparison of percentage difference to neat EP/CF and therefore, the influence of GO within EP/CF-based samples on particle number concentration (C), tensile strength, Young’s modulus, flexural strength and flexural modulus (Note high standard deviations are observed due to the combined deviations of each sample and EP/CF). Table 1. Inferential statistical representation of the particle number concentrations introduced at the peaks due to the drilling on EP/CF-based samples reinforced with GO ( n = 24 for each sample). Lower and upper limits represent the 90% confidence interval on a sampling t-distribution. Table 1. Inferential statistical representation of the particle number concentrations introduced at the peaks due to the drilling on EP/CF-based samples reinforced with GO ( n = 24 for each sample). Lower and upper limits represent the 90% confidence interval on a sampling t-distribution. Sample Mean : X = ( # / cm 3 ) Deviation : S X ¯ ( # / cm 3 ) Minimum (#/cm 3 ) Maximum (#/cm 3 ) 5% Lower Limit of Confidence Interval (#/cm 3 ) 95% Upper Limit of Confidence Interval (#/cm 3 ) EP/CF 2.74 × 10 4 1.81 × 10 4 1.21 × 10 4 6.38 × 10 4 1.84 × 10 4 3.65 × 10 4 EP/CF/GO 0.05 wt.% 5.44 × 10 4 3.30 × 10 4 1.42 × 10 4 11.9 × 10 4 3.79 × 10 4 7.09 × 10 4 EP/CF/GO 0.1 wt.% 3.72 × 10 4 1.39 × 10 4 2.26 × 10 4 6.40 × 10 4 3.03 × 10 4 4.42 × 10 4 EP/CF/GO 0.5 wt.% 9.39 × 10 4 6.59 × 10 4 4.29 × 10 3 18.7 × 10 4 6.10 × 10 4 12.7 × 10 4	https://www.mdpi.com/2073-4433/11/6/573
Usworth (Hylton) (Sunderland) - Airfields of Britain Conservation Trust UK Usworth (Hylton) (Sunderland) About Also known as: Durham / Hylton Aerodrome / RAF Usworth / RAF Hylton / RFC Hylton / Sunderland Airport / Usworth Aerodrome County: Tyne and Wear Current Status: Industry / Museum Date: October 1916 - 31 May 1984 Current Use: Disused Used By: RAF / RFC / FAA (lesser use) / Civil / USAAF (lesser use) Landing Surface Types: Unpaved, later paved Aircraft Roles: Airliner / Bomber / Fighter / General aviation / Trainer Usworth again returned to use in the 1930s. This proved a protracted process as No 607 Squadron of the Auxiliary Air Force formed on 17 March 1930 but did not receive aircraft in the shape of Westland Wapiti day bombers until the end of 1932.The unit later flew Hawker Demons before switching to a fighter role later on in that decade and Gloster Gladiators. Empire Air Days took place annually at Usworth from 1934 until the outbreak of the Second World War, while regular RAF light bomber unit No 103 Squadron also resided at Usworth in peacetime. In terms of appearance the main point of note at this time became the locally famous and unique ‘Beehive’ Lamella hangar that soon became a prominent feature. With the start of the conflict, improvements were made to the airfield, with two hard runways being built. Most of the buildings were situated to the north of the site, in two camps either side of the road. Fighters from Nos 43 and 64 Squadrons operated for short periods in 1940 and into January 1941. Usworth became crucially involved in the Battle of Britain when the Luftwaffe attempted a major second front attack on north-east England on 15 August 1940. No 607 Squadron, which had left its pre-war home for Acklington shortly after war broke out but returned in June 1940 with Hawker Hurricanes, assisted with several other airfields in successfully driving off the attackers. Once No 607 Squadron had again departed in January 1941, in February of that year No 55 Operational Training Unit arrived from Aston Down and training became Usworth’s primary role. Using mainly Hurricanes, this large unit also employed Ouston as a satellite before moving to Annan in the spring of 1942. A few months of Care and Maintenance followed until No 62 OTU formed in June, using Avro Ansons for training radar operators. However, the proximity of a balloon barrage at Sunderland meant that the unit transferred to Ouston in July 1943, essentially bringing to a close flying during the war, aside from some activity from No 31 Gliding School in 1944, which was renamed No 641 GS in 1955. Other subsequent military use around this time notably included No 14 Maintenance Unit employing Usworth from December 1944 to store aero engines. Usworth struggled for some years post-war to establish a more defined role. No 23 Reserve Flying School formed in 1949 to use a variety of aircraft types until disbanding in 1953. Operator of the school, Airwork, also ran No 2 Basic Air Navigation School here from 1951 to 1953. The civilian Newcastle Gliding Club remained as another occasional resident in the 1950s but the RAF finally decided to leave the site in 1958. This was nevertheless far from the end for Usworth as it was bought from the Air Ministry by Sunderland Corporation to become Sunderland Airport. Active again from 1962, beginning its new role with airline charters, the airport continued to be used additionally for general aviation until its closure on 31 May 1984. Sunderland Flying Club had begun operations in 1963 as a major part of this. The site is now a major car manufacturing plant for Nissan, although the North East Land, Sea and Air Museums (NELSAM) provides a welcome reminder of the history of the site at its relocated position to the north of the factory. Sadly the ‘Beehive’ hangar was demolished in 2004 and only tiny scraps of the building remain as a memento. The following organisations are either based at, use and/or have at least potentially significant connections with the airfield (as at 31/07/2018): Nissan Motor Manufacturing (UK) Limited North East Land, Sea and Air Museums (NELSAM) South Hylton Local History Society Washington History Society Notable Past Associated Organisations: Sunderland Corporation Main unit(s) present: No 1 AACU 'G' Flight No 1 Gp Practice Flight No 2 Basic ANS No 2 Civilian Fighter Control Co-operation Unit No 3 Radio Maintenance Unit No 3 Radio Servicing Section No 7 AACU No 14 MU No 20 Initial Training Wing No 23 RFS No 27 GS No 31 EGS / NE31 EGS No 31 GS No 36 Sqn No 43 Sqn No 55 OTU No 60 Initial Training Wing No 62 OTU No 64 Sqn No 72 (Signals) Wing Calibration Flight No 103 Sqn 416th NFS No 607 Sqn No 641 GS No 664 Sqn No 776 Sqn No 1965 Reserve AOP Flight No 2739 Sqn RAF Regiment No 2759 Sqn RAF Regiment Air Crew Disposal Unit Durham UAS Newcastle GC Northern Parachute Centre Ltd Northumbria Parachute Club Sunderland Flying Club Sunderland Parachuting Centre Ltd Tyne-Wear GC Photographs and video from the memorial unveiling at Usworth on 15th September 2018: Pilot Officer Mirosław Orzechowski, one of the Polish pilots serving with No 607 Squadron, standing on a wing of a Hawker Hurricane at RAF Usworth, 14 January 1941. © IWM (HU 128318) Polish pilots serving with No 607 Squadron studying maps on the back wing of a Hawker Hurricane as part of their training at RAF Usworth, 14 January 1941. © IWM (HU 92830) Aerial view of Usworth, c. 1941-1942. © IWM (HU 72106) Kerry Noble landing at Usworth at Sunderland Air Day, June 1971. Courtesy of Kerry Noble Aerial view of Usworth and the Nissan car manufacturing plant, 19 October 2004. The Nissan car manufacturing plant at Usworth, 5 November 2005. Usworth, 1918. Courtesy of Ed Andrews Usworth, 1945. Courtesy of Ed Andrews Usworth, 1945+ with the extended runway. Courtesy of Ed Andrews	https://www.abct.org.uk/airfields/airfield-finder/usworth-hylton-sunderland/
Factors affecting consumer attitude and loyalty: evidence from a Philippine chain of fast-food restaurants’ smart retailing technology \| Request PDF Request PDF \| Factors affecting consumer attitude and loyalty: evidence from a Philippine chain of fast-food restaurants’ smart retailing technology \| Purpose This study aims to explore the influence of perceived value, enjoyment and novelty of fast-food chains’ smart retailing technology (SRT)... \| Find, read and cite all the research you need on ResearchGate Factors affecting consumer attitude and loyalty: evidence from a Philippine chain of fast-food restaurants’ smart retailing technology Journal of Science and Technology Policy Management DOI: 10.1108/JSTPM-09-2022-0159 Abstract Purpose This study aims to explore the influence of perceived value, enjoyment and novelty of fast-food chains’ smart retailing technology (SRT) on Filipino consumers’ attitude and loyalty. Design/methodology/approach Purposive sampling was used in identifying the respondents ( N = 343). The participants were composed of consumers of the leading fast-food chains in the Philippines, where SRT using self-service ordering kiosks is being implemented. The hypotheses were explored using partial least squares path modeling, and predictive-causal was the study’s research design. Findings The results reveal that, among the factors, perceived enjoyment substantially contributes to the formation of favorable consumers’ attitude toward SRT. Moreover, perceived value was found to have a moderate effect on attitude while perceived novelty showed small impact. In terms of consumers’ attitude and loyalty, the two variables were found to have large positive and significant relationship. The moderation analysis shows that consumers’ attitude toward SRT has medium indirect effect on the relationship between perceived enjoyment and loyalty, while there is small indirect influence on the links between perceived value and loyalty, and between perceived novelty and loyalty. Originality/value As more and more fast-food establishments are adopting the use of SRT via self-ordering kiosks, the present study is the only study in the Philippine context that explores how perceived value, enjoyment and novelty affect consumers’ attitude and loyalty. Andrea Sestino Cesare Amatulli Matteo Deangelis This chapter investigates the effectiveness of luxury brands’ messages focused on product sustainability rather than on traditional luxury product features. The study sheds light on the role of perceived product uniqueness and consumers’ conspicuous consumption orientation. An online experiment has been conducted among a sample of 144 participants exposed to a luxury product communication message focused on product sustainability or focused on product performance. Results show that a communication message related to a luxury product and focused on sustainability (vs. performance) leads to higher positive attitudes toward such product and this effect is mediated by consumers’ perceived product uniqueness. Additionally, findings underline the role of conspicuous consumption in magnifying such effect. Implications and suggestions for marketers and managers are discussed. Indah Fatmawati Corporate Social Responsibility (CSR) program has become one of the primary concerns of companies worldwide. For many companies treating the environment and the community well is important to business practice and reputation, and this is reflected in their CSR programs. CSR is a company’s obligation to consider the interests of its employees, customers, shareholders, communities, and the environment and to consider the social and environmental consequences of their business activities. CSR plays an important role in relationship building with customers. Thus, the main purpose of this study is to analyze the relationship between customer perception of CSR and customer trust. The model of this study considers two mediating variables, i.e., company reputation and word of mouth to link CSR to customer trust. This study employs a causal survey design. The respondents were 160 consumers who have purchased products and knew about CSR programs of a global food company. Data analysis was using structural equation modeling (SEM) to test the hypotheses. The results of this study revealed that CSR negatively impacts customer trust. Furthermore, CSR positively impacts corporate study found corporate reputation positively impacts word of mouth and customer trust. While the mediating effect of reputation and word of mouth also positively impacts the relationship between CSR and consumer trust. A good reputation and word of mouth could be connecting buyers and enhances the power of suppliers. © This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nicholas Wilson Keni Keni This study was commenced in order to examine the impact that both perceived usefulness and perceived ease-of-use had toward consumers' loyalty within the computer industry in China, either directly or indirectly through satisfaction and trust. This current study utilized the survey method; questionnaires were selected as the instrument which would be used to collect all of the data from the respondents. All of the questionnaires were electronically distributed through an online survey site to a total of 400 respondents, who originated from, or were living in, five cities in China. After gathering and assessing all of the collected data, a total of 346 items of data were deemed usable, thus enabling these sets of data to be further analyzed by this study. All of these data were further analyzed using the partial least squares-structural equation mod-eling (PLS-SEM) method using SmartPLS 3.3.2 software. Based on the results obtained through the data's analyses, the authors would like to conclude that both perceived usefulness and perceived ease-of-use had a positive and significant impact on customer satisfaction, trust and customer loyalty within China's computer industry. Furthermore, in this study, both customer satisfaction and trust have been found to positively mediate the impact of perceived usefulness and perceived ease-of-use toward customer loyalty; trust serves as a better mediator compared to satisfaction. Meanwhile, this study contributed to the expansion and the enrichment of the literature in the field of marketing by thoroughly examining the implementation of the technology acceptance model (TAM) in predicting customer loyalty within China's computer industry. Influencing Variables Towards the Intention to Purchase (ITP) and Their Research Gaps Purchase intention is important to study because research results are frequently used for making decision about product and services and, the research result could predict future sales for organisation. The objective of the research is to define the independent variables which influence the intention to purchase as the dependent variable. The method used in this study was systematic literature review that was supported by articles from prominent journals. The literature review found that there were contradictions in the relationship between variables, such as variables for intention to purchase (trust, price, perceived value and attitude) and technology management to trust, were contradicting on their hypotheses. The study result provided a significant contribution to the academic field like building a conceptual framework for further study in Asian countries context because there are lack research on Asian contexts about all variables in this research. Besides, the contradiction of all linkages between variables will contribute to the body of knowledge. CSR activities and consumer loyalty: The effect of the type of publicizing medium This research develops a model to predict the effect of advertising a socially responsible activity on perceived corporate social responsibility (CSR) and its influence on consumer loyalty. It examines the relationships between company-cause congruence, corporate credibility, altruism attribution and perceived CSR, and CSR with consumer loyalty, and analyzes the moderating effect of the type of communication medium (i.e. traditional medium vs. social medium). This study is original because it fills the gap in the CSR communication literature in the evaluation of how the use of one or another type of medium to advertise a cause related marketing activity influences the effect of perceived CSR on consumer loyalty. An empirical study was conducted with two samples of consumers, each of which was exposed to the same advertisement, inserted in either a newspaper or posted on a social network. Data were analyzed using structural equation modeling. The results carry implications for CSR activities and communications management as they validate the proposed model that integrates the antecedents of perceived CSR and its influence on loyalty, and show that the traditional medium model has a better fit and its overall effect is greater than the social medium model. From a practical perspective, this study has several implications regarding the importance of communicating CSR activities. CUSTOMERS BEHAVIOUR TOWARDS ONLINE SHOPPING IN INDIA VRShop: A Mobile Interactive Virtual Reality Shopping Environment Combining the Benefits of On- and Offline Shopping In this work, we explored the main characteristics of on- and offline shops with regard to customer shopping behavior and frequency. Thus, we designed and implemented an immersive virtual reality (VR) online shopping environment. We tried to maintain the benefits of online shops, like search functionality and availability, while simultaneously focusing on shopping experience and immersion. By touching the third dimension, VR provides a more advanced form of visualization, which can increase the customer’s satisfaction and thus shopping experience. We further introduced the Virtual Reality Shopping Experience (VRSE) model based on customer satisfaction, task performance and user preference. A case study of a first VR shop prototype was conducted and evaluated with respect to the VRSE model. The results showed that the usability and user experience of our system is above average overall. In summary, searching for a product in a WebVR online shop using speech input in combination with VR output proved to be the best regarding user performance (speed, error rate) and preference (usability, user experience, immersion, motion sickness). Augmenting the eye of the beholder: exploring the strategic potential of augmented reality to enhance online service experiences ko de ruyter Driven by the proliferation of augmented reality (AR) technologies, many firms are pursuing a strategy of service augmentation to enhance customers’ online service experiences. Drawing on situated cognition theory, the authors show that AR-based service augmentation enhances customer value perceptions by simultaneously providing simulated physical control and environmental embedding. The resulting authentic situated experience, manifested in a feeling of spatial presence, functions as a mediator and also predicts customer decision comfort. Furthermore, the effect of spatial presence on utilitarian value perceptions is greater for customers who are disposed toward verbal rather than visual information processing, and the positive effect on decision comfort is attenuated by customers’ privacy concerns. Shopper-Facing Retail Technology: A Retailer Adoption Decision Framework Incorporating Shopper Attitudes and Privacy Concerns Continual innovation and new technology are critical in helping retailers’ create a sustainable competitive advantage. In particular, shopper-facing technology plays an important role in increasing revenues and decreasing costs. In this article, we briefly discuss some of the salient retail technologies over the recent past as well as technologies that are only beginning to gain traction. Additionally, we present a shopper-centric decision calculus that retailers can use when considering a new shopper-facing technology. We argue that new technologies provide value by either increasing revenue through (a) attracting new shoppers, (b) increasing share of volume from existing shoppers, or (c) extracting greater consumer surplus, or decreasing costs through offloading labor to shoppers. Importantly, our framework incorporates shoppers by considering their perceptions of the new technology and their resulting behavioral reactions. Specifically, we argue that shoppers update their perceptions of fairness, value, satisfaction, trust, commitment, and attitudinal loyalty and evaluate the potential intrusiveness of the technology on their personal privacy. These perceptions then mediate the effect of the technology on shopper behavioral reactions such as retail patronage intentions and WOM communication. We present preliminary support for our framework by examining consumers’ perceptions of several new retail technologies, as well as their behavioral intentions. The findings support our thesis that shopper perceptions of the retailer are affected by new shopper-facing technologies and that these reactions mediate behavioral intentions, which in turn drives the ROI of the new technology. The path-to-purchase is paved with digital opportunities: An inventory of shopper-oriented retail technologies TECHNOL FORECAST SOC Malaika Brengman This study focuses on innovative ways to digitally instrument the servicescape in bricks-and-mortar retailing. In the present digital era, technological developments allow for augmenting the shopping experience and capturing moments-of-truth along the shopper's path-to-purchase. This article provides an encompassing inventory of retail technologies resulting from a systematic screening of three secondary data sources, over 2008-2016: (1) the academic marketing literature, (2) retailing related scientific ICT publications, and (3) business practices (e.g., publications from retail labs and R&D departments). An affinity diagram approach allows for clustering the retail technologies from an HCI perspective. Additionally, a categorization of the technologies takes place in terms of the type of shopping value that they offer, and the stage in the path-to-purchase they prevail. This in-depth analysis results in a comprehensive inventory of retail technologies that allows for verifying the suitability of these technologies for targeted in-store shopper marketing objectives (cf. the resulting online faceted-search repository at . www.retail-tech.org). The findings indicate that the majority of the inventoried technologies provide cost savings, convenience and utilitarian value, whereas few offer hedonic or symbolic benefits. Moreover, at present the earlier stages of the path-to-purchase appear to be the most instrumented. The article concludes with a research agenda. Is the smartwatch an IT product or a fashion product? A study on factors affecting the intention to use smartwatches Smartwatches, among many wearable form factors, have become an important starting point for the wide dissemination of wearable devices. Not only are smartwatches a new IT product, they are also a fashion product. Recognizing the smartwatch as the converging point of IT innovation and fashion, the current study examines whether factors germane to the characteristics of fashion products affect the intention to use smartwatches. Utilizing the technology acceptance model (TAM) as the base framework, the current study extended the model by incorporating perceived enjoyment and perceived self-expressiveness, which are influenced by an individual’s vanity and need for uniqueness. The findings from 562 Korean respondents indicated that the characteristics of smartwatches as fashion products significantly explain the intention to use a smartwatch, particularly the individual’s desire for uniqueness. A limited effect of vanity on self-expressiveness implies that the smartwatch is not yet deemed a luxury commodity. Theoretical and practical implications, as well as the limitations of the study are further discussed. Applying configurational analysis to IS behavioural research: a methodological alternative for modelling combinatorial complexities Hongxiu Li Jun 2020 IND MANAGE DATA SYST Wynne W. Chin Cheah Jun Hwa Yide Liu Tat-Huei Cham Purpose Partial least squares structural equation modeling (PLS-SEM) has become popular in the information systems (IS) field for modeling structural relationships between latent variables as measured by manifest variables. However, while researchers using PLS-SEM routinely stress the causal-predictive nature of their analyses, the model evaluation assessment relies exclusively on criteria designed to assess the path model's explanatory power. To take full advantage of the purpose of causal prediction in PLS-SEM, it is imperative for researchers to comprehend the efficacy of various quality criteria, such as traditional PLS-SEM criteria, model fit, PLSpredict, cross-validated predictive ability test (CVPAT) and model selection criteria. Design/methodology/approach A systematic review was conducted to understand empirical studies employing the use of the causal prediction criteria available for PLS-SEM in the database of Industrial Management and Data Systems (IMDS) and Management Information Systems Quarterly (MISQ). Furthermore, this study discusses the details of each of the procedures for the causal prediction criteria available for PLS-SEM, as well as how these criteria should be interpreted. While the focus of the paper is on demystifying the role of causal prediction modeling in PLS-SEM, the overarching aim is to compare the performance of different quality criteria and to select the appropriate causal-predictive model from a cohort of competing models in the IS field. Findings The study found that the traditional PLS-SEM criteria (goodness of fit (GoF) by Tenenhaus, R2 and Q2) and model fit have difficulty determining the appropriate causal-predictive model. In contrast, PLSpredict, CVPAT and model selection criteria (i.e. Bayesian information criterion (BIC), BIC weight, Geweke–Meese criterion (GM), GM weight, HQ and HQC) were found to outperform the traditional criteria in determining the appropriate causal-predictive model, because these criteria provided both in-sample and out-of-sample predictions in PLS-SEM. Originality/value This research substantiates the use of the PLSpredict, CVPAT and the model selection criteria (i.e. BIC, BIC weight, GM, GM weight, HQ and HQC). It provides IS researchers and practitioners with the knowledge they need to properly assess, report on and interpret PLS-SEM results when the goal is only causal prediction, thereby contributing to safeguarding the goal of using PLS-SEM in IS studies. Understanding the attitude and intention to use smartphone chatbots for shopping May 2020 Tech Soc Dharun Kasilingam Using the technology acceptance model and diffusion of innovations theory, this study evaluated the intention of consumers to use chatbots on smartphones for shopping. Chatbot is a relatively new technology and is expected to dominate mobile commerce and shopping applications in future. Hence, this study aimed to determine the association of perceived usefulness, perceived ease of use, perceived enjoyment, price consciousness, perceived risk, trust, and personal innovativeness with attitude and intention to use chatbots for shopping. Respondents were asked to fill a questionnaire after using a Facebook e-commerce chatbot that was specifically created for this study. In total, 350 responses were analyzed using partial least squares structural equation modeling. Results indicated that attitude toward chatbots was considerably influenced by the variables perceived usefulness, perceived ease of use, perceived enjoyment, price consciousness, perceived risk, and personal innovativeness. However, intention to use was directly influenced only by trust, personal innovativeness, and attitude. Mediation analysis indicated that full mediation occurs through the attitude variable for most direct relationships. Moderation analysis by using age, gender, and prior experience with mobile shopping applications indicated considerable differences between the groups in terms of the strength of certain relationships and the mean responses between the variables. Examining the antecedents and consequences of perceived shopping value through smart retail technology Jan 2020 J Retailing Consum Serv Sujana Adapa Syed Muhammad Fazal e Hasan M S Balaji Gary Mortimer This study examines the antecedents and consequences of perceived shopping value through smart retail technology. Specifically, we propose that perceived complexity, perceived advantage, perceived novelty and perceived risk of using smart retail technologies determine consumers’ perceived shopping value, which, in turn, influences their store loyalty and intentions to adopt smart retail technology. Further, we propose that consumer innovativeness moderates these relationships. Survey responses from 338 actual shoppers with prior experience of smart retail technology were used to test the research model, while structural equation modelling was used to develop the proposed hypotheses. The present study offers a better understanding of consumer adoption of smart retail technology that may help managers to develop adoption strategies for successful implementation of smart retail technology in-store. Mirror, mirror on the wall, are we ready for Gen-Z in Marketplace? A study of Smart Retailing Technology in Malaysia Nov 2019 Siew Imm Ng Jo Ann Ho Lim Xin Jean In this digitalization age, smart technologies are on the cusp of changing all business sector including retailing. Today’s consumers desire to shop in a smart store where the physical products on display are connected to the Internet world. This study proposes a model for investigating Gen-Z consumers’ expectations towards the smart retail technology in the Malaysian context through the application of the Stimulus-Organism-Response framework. The findings of this study provide beneficial insights for retailer who desire to pursue a smart-related acquisition strategy. Retailers are recommended to embrace on three key features: innovative solution (high usefulness), reliable benefits (high perceived value) and fun experience (high enjoyment) in order to provide Gen-Z consumers with compelling experiences. Additionally, retailers are suggested to acknowledge the importance of managing consumers’ attitude in driving positive WOM evaluations. Online grocery shopping in Thailand: Consumer acceptance and usage behavior J Retailing Consum Serv Fabian Driediger Veera Bhatiasevi This study is the first of its kind to examine the acceptance and usage behavior of online grocery shopping in Thailand. It proposes an extension of the technology acceptance model, including subjective norm, visibility, perceived risk, and perceived enjoyment to better understand the factors and the extent to which they lead to the acceptance or rejection of online grocery shopping. In order to accurately test the variables and the relationships of the proposed research model, a questionnaire was developed and distributed to 450 residents in the Bangkok area, of which 263 valid responses were returned back to the researcher. Instrument development was done by using existing scales and items from the current literature on technology acceptance. Partial least squares structural equation modeling (PLS-SEM) was used for this research, with the results emphasizing that perceived ease of use, perceived usefulness, intention to use, subjective norm, and perceived enjoyment have a statistically significant relationship towards the acceptance of online grocery shopping in Thailand. By contrast, visibility and perceived risk were found to have no significant impact on the perceived usefulness of online grocery shopping. The results and implications are summarized in the discussion part of this paper where valuable recommendations for decision makers are provided. View A multi-analytical approach to peer-to-peer mobile payment acceptance prediction Apr 2019 J Retailing Consum Serv Zoran Kalinić Veljko Marinković Francisco Liébana-Cabanillas Sebastian Molinillo (Manuscript available at https://authors.elsevier.com/c/1YqEu3SU~VaAVN). This research is a pioneering study into peer-to-peer mobile payment (P2PM-pay) systems' adoption. It proposes a behavioral model of the use of P2PM-pay systems and identifies the key antecedents of the customer's intention to use. Using a two-stage approach, the research model is assessed with data collected through an online survey from a sample of 701 respondents. In the first step, structural equation modeling (SEM) is used to determine P2P mobile payment acceptance predictors. In the second step, neural network models are used to rank the relative influence of significant predictors obtained from the SEM. The results show that consumers perceive the usefulness of P2PM-pay as the most important factor influencing their decision to adopt this innovative technology. The significant impact of social norms and perceived trust are also corroborated. The paper provides important strategic guidelines for the management of companies involved in the development and implementation of P2PM-pay systems. Understanding the diffusion of virtual reality glasses: The role of media, fashion and technology TECHNOL FORECAST SOC Marc Florian Herz Philipp A. Rauschnabel Facebook, Google and many other established players’' future will encompass a new media format - Virtual Reality (VR) - that require novel devices such as VR glasses. While market forecasts are promising, recent diffusion rates indicate that consumer acceptance is still limited. By incorporating existing and proposing new benefits and risks through the lenses of media, technology, and fashion research, the authors develop and test a comprehensive framework to study consumer reactions to wearable VR glasses. Results of an empirical study indicate various novel and interesting findings. For example, health and privacy risks diminish adoption rates, whereas – contrary to other technologies – psychological or physical risks – do not. Likewise, fashionable designs and wearable comfort – two novel constructs investigated in this research – matter in addition to established utilitarian and hedonic constructs. Finally, this study includes a novel perspective on media technologies in by showing that adoption intention is highest when consumers expect to experience both a strong sense of virtual embodiment (the sensation of being another person) and virtual presence (the sensation of being at another place), while the presence of only one of these conditions might even have a negative effect. Consumer Resistance to Innovation in Services: Challenges and Barriers in the Internet of Things Era Article Zied Mani Ines Chouk The Internet of Things is expected to be the next phase of the Internet revolution and to transform consumers' service experience. It nevertheless raises challenges on innovation in services. Based on the Ram and Sheth model (1989), this work attempts to provide a better understanding of the barriers that lead to consumers' resistance to smart services as an innovation. To this end, our research adopts an integrative framework that combines functional barriers, psychological barriers and individual barriers to explain consumer resistance to smart services. Structural equation modelling was used to test this theoretical framework. Our research enriches the existing literature by (1) adapting the Ram and Sheth model (1989) to the evolution of digital technologies (technological vulnerability barriers), (2) taking into account the ideological aspect of resistance (ideological barriers) and (3) considering dispositional variables (individual barriers). Moreover, it highlights the key role of skepticism towards IoT devices as a mediator between technological vulnerability barriers and individual barriers on the one hand and consumer resistance to smart services on the other. This article is protected by copyright. All rights reserved. Predictors of customer acceptance of and resistance to smart technologies in the retail sector May 2018 J Retailing Consum Serv Ali Quazi Mohammed Quaddus Sanjit K. Roy M S Balaji In recent decades, rapid advances in Internet technology have led to numerous innovative smart technologies. This research investigates the customer acceptance of and resistance to smart technologies in the retail sector, by integrating the literature on technology acceptance model, system characteristics, technology readiness, and store reputation. Data were collected using a quantitative survey and analysed using symmetrical PLS path modelling and asymmetrical fuzzy set qualitative comparative analysis (fsQCA). Results show complex relationships among perceived technology readiness, perceived ease of use, perceived usefulness, superior functionality, perceived adaptiveness, and store reputation in determining customers’ attitudes and behavioural intentions towards smart retail technologies. The findings also show that technology readiness does not directly affect customer attitude but does indirectly through perceived innovation characteristics. The findings indicate that retail stores should focus on smart technologies that are simple, yet offer enhanced customer value through improved shopping efficiency. Findings also suggest that retail stores can engage in brand management strategies to improve customers’ acceptance of smart technologies. Store environment and consumer purchase behavior: Mediating role of consumer emotions Jul 1997 PSYCHOL MARKET Elaine Sherman Anil Mathur	https://www.researchgate.net/publication/370122409_Factors_affecting_consumer_attitude_and_loyalty_evidence_from_a_Philippine_chain_of_fast-food_restaurants%27_smart_retailing_technology
Genesis 3:16 (KJV) - Forerunner Commentary Unto the woman he said, I will greatly multiply thy sorrow and thy conception; in sorrow thou shalt bring forth children; and thy desire shall be to thy husband Genesis 3:16 ( King James Version ) A.F.VA.S.V.Amplified®Darby K.J.V.N.A.S.B.NASB E-PrimeR.S.V.Young'sCompare all Book Notes Barnes' Book Notes Jamieson, Fausset, and Brown Book Notes Robertson's Book Notes (NT) Commentaries Adam Clarke Barnes' Notes Forerunner Commentary Jamieson, Fausset, and Brown John Wesley's Notes Matthew Henry People's Commentary (NT) Robertson's Word Pictures (NT) Scofield Definitions Interlinear Library Topical Studies X-References Commentaries: Forerunner Commentary What is the Forerunner Commentary? Genesis 3:16 The first curse includes the whole processes of childbearing, from conception to birth. The Hebrew word rendered "conception" in the New King James version (NKJV) includes the entire pregnancy, while "bring forth" can mean both the beginning or end of the birth process. The Revised Standard Version translates these clauses as, "I will greatly multiply your pain in childbearing; in pain you shall bring forth children." A human female is unique among mammalian creatures in this respect. Animal females generally bear their young without pain and rarely sicken and die during or from the experience. Women, on the other hand, always experience pain and grief throughout their pregnancies—from morning sickness to contractions—and have historically had a very high mortality rate from childbirth. Better nutrition and hygiene have cut the numbers of deaths dramatically, but the pain and grief remain. Fortunately,Godis a God of mercy. He put within the human female the ability to "forget" her pains in childbirth soon thereafter.JesusHimself mentions this inJohn 16:21: A woman, when she is in labor, has sorrow because her hour has come; but as soon as she has given birth to the child, she no longer remembers the anguish, forjoythat a human being has been born intothe world. This curse on Eve has a direct relationship with the end of the curse on the serpent, which involves the woman's "seed," both general and specific (Genesis 3:15). We can infer that God intends us to understand that, because ofsin, producing "seed" to fightSatanand his seed will be made more difficult. In a spiritual sense, the church, "the mother of us all," endures great hardship in producing children of God. Thus, the Bible testifies, "thekingdom of heavensuffers violence, and theviolent take it by force" (Matthew 11:12), "We must through many tribulations enter thekingdom of God" (Acts 14:22), and "all who desire to live godly in Christ Jesus will suffer persecution" (II Timothy 3:12). Even the sinless Christ, the promised Seed, was "a Man of sorrows and acquainted with grief" (Isaiah 53:3), forced by sin—yet willing—to bear the agonies of human life and death to become the Son of God, the Firstborn among many brethren. Richard T. RitenbaughThe First Prophecy (Part Two) Genesis 3:16-17 Eve's curse has resulted in the virtual subjugation of women ever since. Until the Enlightenment in the 18th century, women had few rights, if any. Fathers sold their daughters into slavery or wed them to the highest bidder. Wives existed to give the husband pleasure and sons and to keep the house. Many societies insisted that women be veiled in public, and some considered it a criminal act for a woman to walk out of her house without a chaperone. In short, a woman was chattel. Not every society was this strict. Because ofGod's law, Israel was one of the most enlightened in this area. Israelite women had certain rights of inheritance, and they could even own land and run businesses (Proverbs 31:16, 24), situations unheard of in other nations. Deborah, an ancient Margaret Thatcher, judged Israel and gave her people forty years ofpeace(Judges 4:4;5:31). An Israelite woman's life has frequently been better than her Gentile counterpart's because of Israel's acquaintance with the Bible. Richard T. RitenbaughA Woman's World Related Topics: Curse on Eve \| Eve's Curse \| Subjugation of Women \| Woman's Curse \| Women in Authority \| Women's Rights \| Women's Role \| Women, Subjugation of Genesis 3:14-19 The Bible's first prophecy contains three major curses, one each uponSatan, women, and men. Or does it? The answer depends on one's perspective and time reference. Certainly, the curse on Satan seems a mixed bag! Men have either been subject to his deceptions or fiercely waging war with him for six thousand years. Yet it is the struggle of the fight that prepares our character to inherit eternal life. We live in hope andfaiththatGodwill see the curse through to its end, the total humiliation and imprisonment of theAdversary. To a woman in labor or to a man sweating out in a field under the sun, God's pronouncements surely feel like curses. Yet, maybe only moments later, the satisfaction andjoyin seeing a healthy baby or a job well done can make it all seem worthwhile. We feel grateful that God has given us such blessings. If nothing else, this should make us think about the "curses" and "blessings" in our lives. Could something terrible turn out for the best? Could seeing "our ship come in" prove our ruin? There is much more to God's gifts and judgments than meets the eye: Oh, the depth of the riches both of thewisdomand knowledge of God! How unsearchable are His judgments and His ways past finding out! "For who has known the mind of the L ORD? Or who has become His counselor?" "Or who has first given to Him and it shall be repaid to him?" For of Him and through Him and to Him are all things, to whom be glory forever. Amen. (Romans 11:33-36) Richard T. RitenbaughThe First Prophecy (Part Three) Genesis 3:16-19 The matter of leadership—whether nationally, locally, at home, on the job, or on the team—has always been a vexing problem for mankind. “Always” should be taken literally becauseGenesis 3:16-19reveals it was a major part of several issues that triggered mankind's circumstances ever since, down to this very second. After Adam's and Eve's sins,Godimposed curses, at the same time pointedly stating why this major flaw in man's character helped to trigger the human condition that persists today. Notice that God mentions to Adam, whom He had appointed as leader of the family through which He intended to populate the entire earth, “You have heeded the voice [counsel] of your wife.” In other words, he had failed to lead the only person who was then under his authority. He took her counsel rather than do what God had commanded him to do, and thus he sinned. The context does not state why he did so, but what resulted was an act of idolatry. He put her counsel before God's, breaking the first commandment. How long did Adam ponder the challenge of the serpent's arguments to break God's commandment by eating of the Tree of the Knowledge of Good and Evil? A few seconds? A few minutes? Whatever it was, in comparison to the amount of time that has passed since, it remains as little more than a flash of lightning. Yet, consider how this seemingly minorsinmotivated God to react. This singular episode in Eden illustrates how seriously God treats sin as compared to how lightly we tend to take it. It was a brief moment in time, when Adam, along with Eve—only two people—failed to exercise leadership by obeying God's simple stricture. What they chose to do instead has brought far more difficult lives on billions of people—difficulty that otherwise may have never occurred. Life, righteousness, and sin do not operate in a vacuum. There is no such thing as sin that does not hurt others, as some so foolishly think or proclaim to justify themselves. Ecclesiastes 7:29makes a telling statement regarding our creation: “Truly this only have I found: That God made man upright, but they have sought out many schemes.” Solomon intends that we understand Adam and Eve to be representative of all mankind. However, Genesis 3 makes clear that they exercised their leadership by leading us into sin. In so doing, they led us all to fall from the pinnacle of human innocence in which they had been created. John W. RitenbaughLeadership and Covenants (Part One) Genesis 3:7-21 Here we have the Bible's first sermon. This is what Abel heard, believed, and submitted to. The same instruction merely informed Cain. Adam and Eve were the first sinners to stand beforeGodand be called into account. In this passage are four elements that apply to what Abel believed. The first element is that, in order for a sinner to stand before God, nakedness must be covered. Nakedness, both spiritual and physical, has wide usage as a symbol. At its best, it indicates innocence, child-like simplicity, and vulnerability. At its worst, it indicates humiliation, guilt, shame, and punishment. Adam and Eve were attempting to hide their humiliation, guilt, and shame when they grabbed a few fig leaves to provide covering. An interesting spiritual lesson comes in understanding an application of the symbolism here. Adam and Eve threw together as a covering whatever was handy at the moment. What they chose to cover themselves with physically was totally inadequate as a spiritual covering. God immediately rejected their effort, which is the main instruction of this vignette. A secondary teaching is that many carnal people today think it does not matter what they physically wear when they come before God at church services. Oh, yes, it does! These days, people arrive at church to worship wearing all kinds of casual clothing. In fact, many churches invite them to do so, advertising themselves as "casual"! Sometimes this reflects a matter of ignorance; they just do not know any better. At other times, it reveals a serious matter of disrespect for the primary covering—Christ's sacrifice, as we shall see shortly. It is good to remember the overall principle to appear before God covered with acceptable covering. The symbolic instruction carries through to both physical and spiritual applications, and the person who cares what God thinks will do his best to conform to Him. God covered Adam and Eve with truly fine clothing. That is our example. The second element Genesis 3 reveals takes us a step further spiritually in regard to the covering: What humans devise in terms of covering spiritual nakedness is, in reality, worthless. The third element clarifies this further: God Himself must supply the only covering that is spiritually adequate. The fourth element is that the only adequate spiritual covering is by means of death. As in the first element, there are two lines of instruction. The first leads to the necessity of the second, if life is to continue. The wages ofsinis death (Romans 6:23). The underlying principle is that we are always to give of our best to the Master. When we fail, the death penalty is imposed. This, then, brings forth a second teaching: In a spiritual sense, the entire human race sinned in Adam and Eve, who represented all mankind at the time. Since the wages of sin is death, and all have subsequently sinned, all of us must receive that wage—or another, an innocent One on whom death has no claim because He never sinned, must substitute for us. However, we find it clearly spelled out in Romans that there must be a link between us and the Substitute (Romans 4:1-4, 11-12, 16, 19-20, 23-25; 5:1-2). Faithin the sacrifice ofJesus Christis the link between us and God's forgiveness, which provides the acceptable spiritual covering necessary to be received into God's presence and receive the gift of life. The second aspect of the fourth element also involves another death—ours. In this case, it is not a literal death but a spiritual one: What shall we say then? Shall we continue in sin that grace may abound? Certainly not! How shall we who died to sin live any longer in it? . . . knowing this, that our old man was crucified with Him, that the body of sin might be done away with, that we should no longer be slaves of sin. For he who has died has been freed from sin. Now if we died with Christ, we believe that we shall also live with Him. (Romans 6:1-2, 6-8) This death is achieved throughrepentancebecause one believes he is a sinner in need of God's forgiveness, having broken His law and earned death. What we have just reviewed must have been taught to Cain and Abel, probably by Adam. How do we know this? BecauseHebrews 11:4tells us that Abel offered by faith, and faith comes by hearing. He heard the divine words given by God to Adam and Eve, which were passed to him, and Abel believed. Cain heard the same words, but did not believe as Abel did. More proof is recorded following Cain's rejection. God says to him inGenesis 4:7, "If you do well, will you not be accepted? And if you do not do well, sin lies at the door. And its desire is for you, but you should rule over it." God clearly indicates a choice between right and wrong. Good and evil faced Cain and Abel. The one brother by faith chose what was right in God's eyes, while the other chose what was right in his own eyes. In essence, he chose death. John W. RitenbaughThe Christian Fight (Part Three) Genesis 3:16 The curse is in two parts, each composed of two parallel clauses. The first part deals with childbearing and the second with marital relations. With two quick strokesGodillustrates the bane of women throughout the ages. On the surface, this verse seems fairly straightforward. However, the word-for-word translation obscures a great deal of its meaning. Because the Hebrew wording includes so much more than the words' literal meanings, both curses give translators fits. They do not want to stray too far from God's exact words, nor do they wish to leave out underlying ideas expounded by Paul in the New Testament. In the end, most choose to translate the passage word for word. God's pronouncement on Eve stands in stark contrast to the positive tone He had given to childbearing and marriage in earlier chapters. He expresses His command inGenesis 1:28in glowing terms: "Then God blessed them, and God said to them, 'Be fruitful and multiply; fill the earth and subdue it.'" Likewise,Genesis 2:18, 24 paints a positive picture of a woman's role in marriage: And the Lord God said, "It is not good that man should be alone; I will make him a helper comparable to him." . . . Therefore a man shall leave his father and mother and be joined to his wife, and they shall become one flesh. Whensinbecomes a factor, however, childbearing and marriage lose their God-intended luster, and if human nature takes its course, pain, suffering, and bitter subjection are inevitable. Richard T. RitenbaughThe First Prophecy (Part Two) Genesis 3:16 The second of Eve's curses deals with her relationship with her husband. It explains why many marriages fail and why many of the rest are unhappy. As mentioned before, human relationships are just as likely to fail as to succeed when men and women rely on human knowledge rather than revealed, godlywisdom. The NKJV's rendering of the latter half ofGenesis 3:16is typical of many translations: "Your desire shall be for your husband, and he shall rule over you." From this we can see that the two clauses cannot be parallels. Instead, they form a statement of action and reaction. Because the woman "desires" her husband, he will "rule over" her. Yet this does not make much sense as a curse. Why should a woman's desire for her husband cause him to dominate her? Most men would gladly accept his wife's desires for him, causing him to treat her more gently rather than roughly, as is implied in this verse. How are we to understand this? The key is in the word "desire," translated from the Hebrew tesuqah, which the Brown, Driver and Briggs lexicon calls "unusual and striking" (p. 1003). It occurs only three times in theOld Testament: here,Genesis 4:7, andSong 7:10. It can carry the sense of sexual longing (as in the Song of Songs), but its usage inGenesis 4:7shows another side, that of a desire to overcome or defeat another: "[Sin's] desire is for you, but you should rule over it." This latter meaning fitsGenesis 3:16better than the former. Thus,Godis saying that a woman's desire will be to gain the upper hand over her husband, but because she is the weaker vessel, her husband will put her down by force, if need be. The curse is that, in the main, women will lose the battle of the sexes. History bears this out. Until the advent of women's rights movements, women were virtually their husband's property, treated as heir-producing machines, given little freedom, and forced to serve their husband's every whim. In many cultures, men bought and sold women like cattle. Some cultures maintain this custom even today. Only where true Christianity flourishes is there any real easing of this curse.Ephesians 5:22-33teaches how we can decrease its effects within our marriages—by emulating the virtues of Christ's relationship with the church. Thus, wives are told to submit rather than contend, and husbands are commanded toloverather than dominate. It takes conscious effort to overcome the evil, ingrained habits of 6,000 years of misguided practice. Richard T. RitenbaughThe First Prophecy (Part Two) Genesis 3:14-19 Often neglected in favor of more "exciting" prophecies, this first prophecy holds the fundamental principles for understanding the nature of Satan's relationship toChristand the church, woman's relationship with man, man's relationship with nature, and sin's role in human suffering. Few subjects are more important! The setting of this prophecy provides the necessary background information we need to understand the full implications of God's pronouncements in these verses. Adam and Eve were still living in the Garden of Eden.Satan, speaking through a serpent, had just deceived Eve into eating the fruit of the tree of the knowledge of good and evil. She, in turn, had persuaded Adam to do the same. These sins demanded the judgment ofGod, which He expresses as curses that would result from their disobedience. At first glance, the curses seem severe. These two innocents—babes, really—had no armor "against the wiles of the devil" (Ephesians 6:11). However, they had received instruction from God on the very point in question (Genesis 2:16-17), and this should have been sufficient to deter them. From God's point of view, their actions were sheer rebellion! In addition, when God inquired about their actions (Genesis 3:11), they neither admitted their transgressions nor sought forgiveness. Instead, they shifted the blame—Adam to Eve, and Eve to Satan (verses 12-13)! Their actions throughout this scenario told God plenty about their character, making his predictions certain. Thus, what we see is that God did not curse them—they cursed themselves! Because of sin's predictable course, God merely voices the consequences of their actions in prophetic terms. This prophecy, then, includes Satan's ultimate guilt and punishment, mankind's battle of the sexes and struggle to survive, and the need for a Savior to repair the damage they had caused. What we see in microcosm is the plan of God! Richard T. RitenbaughThe First Prophecy (Part One)	https://www.bibletools.org/index.cfm/fuseaction/Bible.show/sVerseID/72/eVerseID/73/
ute significantly to 5-HT-induced excitatory effects in the PFC of young adult rats. This is consistent with the results obtained in previous studies of adult rat PFC (\u003Ca id=\u0022xref-ref-4-2\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-4\u0022\u003EAraneda and Andrade, 1991\u003C\/a\u003E; \u003Ca id=\u0022xref-ref-67-3\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-67\u0022\u003ETanaka and North, 1993\u003C\/a\u003E).\u003C\/p\u003E\u003C\/div\u003E\u003Cdiv id=\u0022sec-7\u0022 class=\u0022subsection\u0022\u003E\u003Ch3\u003EPostsynaptic mechanisms are responsible for 5-HT-induced\u00a0excitation\u003C\/h3\u003E\u003Cp id=\u0022p-27\u0022\u003EIn pyramidal neurons of the adult PFC, activation of 5-HT\u003Csub\u003E2A\u003C\/sub\u003E receptors produces a substantial increase in spontaneous excitatory synaptic transmission (\u003Ca id=\u0022xref-ref-1-1\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-1\u0022\u003EAghajanian and Marek, 1997\u003C\/a\u003E; \u003Ca id=\u0022xref-ref-35-1\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-35\u0022\u003ELambe et al., 2000\u003C\/a\u003E). Therefore, I examined the contribution of presynaptic and postsynaptic effects to 5-HT-induced excitatory responses in neurons aged P10\u2013P12. As illustrated in Figure \u003Ca id=\u0022xref-fig-5-1\u0022 class=\u0022xref-fig\u0022 href=\u0022#F5\u0022\u003E5\u003C\/a\u003E\u003Cem\u003EA\u003C\/em\u003E, 5-HT (10 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) elicited an inward current at \u221274 mV (\u221228 \u00b1 4 pA; \u003Cem\u003En\u003C\/em\u003E = 6), accompanied by a substantial increase in noise. After blocking synaptic transmission with ACSF containing 0 Ca\u003Csup\u003E2+\u003C\/sup\u003E (nominal) and 8 m\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E Mg\u003Csup\u003E2+\u003C\/sup\u003E, 5-HT (10 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) induced an inward current (\u221234 \u00b1 8 pA; \u003Cem\u003En\u003C\/em\u003E = 4) (Fig. \u003Ca id=\u0022xref-fig-5-2\u0022 class=\u0022xref-fig\u0022 href=\u0022#F5\u0022\u003E5\u003C\/a\u003E\u003Cem\u003EB\u003C\/em\u003E) with little increase in noise. Similar results were obtained when TTX (0.3 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) was included in the ACSF (\u221233 \u00b1 4 pA; \u003Cem\u003En\u003C\/em\u003E = 16) (Fig. \u003Ca id=\u0022xref-fig-5-3\u0022 class=\u0022xref-fig\u0022 href=\u0022#F5\u0022\u003E5\u003C\/a\u003E\u003Cem\u003EC\u003C\/em\u003E). These results suggest that the inward current is the result of a direct postsynaptic action of 5-HT on these neurons, whereas the increase of noise is primarily attributable to presynaptic effects. As shown in previous studies (\u003Ca id=\u0022xref-ref-1-2\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-1\u0022\u003EAghajanian and Marek, 1997\u003C\/a\u003E; \u003Ca id=\u0022xref-ref-76-1\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-76\u0022\u003EZhou and Hablitz, 1999\u003C\/a\u003E), this increase of spontaneous EPSCs was blocked by the antagonists of glutamate receptors, DNQX (20 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) and\u003Cspan class=\u0022sc\u0022\u003Ed\u003C\/span\u003E-APV (50 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E;\u003Cem\u003En\u003C\/em\u003E = 5; data not shown). However, 5-HT-induced excitatory responses persisted in the presence of DNQX and\u003Cspan class=\u0022sc\u0022\u003Ed\u003C\/span\u003E-APV (\u003Cem\u003En\u003C\/em\u003E = 5) (Fig.\u003Ca id=\u0022xref-fig-5-4\u0022 class=\u0022xref-fig\u0022 href=\u0022#F5\u0022\u003E5\u003C\/a\u003E\u003Cem\u003ED\u003C\/em\u003E). Together, these results suggest that the excitation induced by 5-HT is mediated primarily via postsynaptic mechanisms.\n\u003C\/p\u003E\u003Cdiv id=\u0022F5\u0022 class=\u0022fig pos-float odd\u0022\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F5.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Postsynaptic mechanisms mediate 5-HT-induced excitatory effects. A, Whole-cell currents induced by 5-HT (10 \u0026#x3BC;m) in a P10 neuron voltage-clamped at \u0026#x2212;74 mV. HP, Holding potential. B, 5-HT-induced whole-cell currents in ACSF containing 0 Ca2+ and 8 mm Mg2+ in a P10 neuron.C, 5-HT-induced whole-cell currents in the presence of TTX (0.3 \u0026#x3BC;m) in a P10 neuron. D, 5-HT-induced excitatory effects in the presence of DNQX andd-AP5 in a P9 neuron.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-1200318417\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;Postsynaptic mechanisms mediate 5-HT-induced excitatory effects. \u0026lt;em\u0026gt;A\u0026lt;\/em\u0026gt;, Whole-cell currents induced by 5-HT (10 \u0026#x3BC;m) in a P10 neuron voltage-clamped at \u0026#x2212;74 mV. HP, Holding potential. \u0026lt;em\u0026gt;B\u0026lt;\/em\u0026gt;, 5-HT-induced whole-cell currents in ACSF containing 0 Ca\u0026lt;sup\u0026gt;2+\u0026lt;\/sup\u0026gt; and 8 mm Mg\u0026lt;sup\u0026gt;2+\u0026lt;\/sup\u0026gt; in a P10 neuron.\u0026lt;em\u0026gt;C\u0026lt;\/em\u0026gt;, 5-HT-induced whole-cell currents in the presence of TTX (0.3 \u0026#x3BC;m) in a P10 neuron. \u0026lt;em\u0026gt;D\u0026lt;\/em\u0026gt;, 5-HT-induced excitatory effects in the presence of DNQX andd-AP5 in a P9 neuron.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F5.medium.gif\u0022 width=\u0022190\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F5.medium.gif\u0022 width=\u0022190\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F5.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 5.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F5.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/452157\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 5.\u003C\/span\u003E \u003Cp id=\u0022p-28\u0022 class=\u0022first-child\u0022\u003EPostsynaptic mechanisms mediate 5-HT-induced excitatory effects. \u003Cem\u003EA\u003C\/em\u003E, Whole-cell currents induced by 5-HT (10 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) in a P10 neuron voltage-clamped at \u221274 mV. HP, Holding potential. \u003Cem\u003EB\u003C\/em\u003E, 5-HT-induced whole-cell currents in ACSF containing 0 Ca\u003Csup\u003E2+\u003C\/sup\u003E and 8 m\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E Mg\u003Csup\u003E2+\u003C\/sup\u003E in a P10 neuron.\u003Cem\u003EC\u003C\/em\u003E, 5-HT-induced whole-cell currents in the presence of TTX (0.3 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) in a P10 neuron. \u003Cem\u003ED\u003C\/em\u003E, 5-HT-induced excitatory effects in the presence of DNQX and\u003Cspan class=\u0022sc\u0022\u003Ed\u003C\/span\u003E-AP5 in a P9 neuron.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cdiv id=\u0022sec-8\u0022 class=\u0022subsection\u0022\u003E\u003Ch3\u003EMultiple membrane conductances are involved in 5-HT-induced\u00a0excitation\u003C\/h3\u003E\u003Cp id=\u0022p-29\u0022\u003ETo determine membrane conductances involved in 5-HT-induced postsynaptic responses, I made voltage-clamp recordings from neurons aged between P10 and P14. TTX (0.3 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) was present throughout the recording to block voltage-gated Na\u003Csup\u003E+\u003C\/sup\u003E channels and 5-HT-induced presynaptic effects (\u003Ca id=\u0022xref-ref-1-3\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-1\u0022\u003EAghajanian and Marek, 1997\u003C\/a\u003E; \u003Ca id=\u0022xref-ref-76-2\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-76\u0022\u003EZhou and Hablitz, 1999\u003C\/a\u003E). Input resistance was measured by stepping from \u221260 to \u221290 or \u2212120 mV. 5-HT (10 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) produced no significant change in input resistance of neurons (2.1 \u00b1 1.4%; \u003Cem\u003En\u003C\/em\u003E = 16;\u003Cem\u003Ep\u003C\/em\u003E \u0026gt; 0.1). To examine the \u003Cem\u003EI\u003C\/em\u003E\u2013\u003Cem\u003EV\u003C\/em\u003Erelationship of 5-HT-induced inward current, slow voltage ramps from \u2212145 to \u221255 mV were applied before, during, and after 5-HT application (Fig. \u003Ca id=\u0022xref-fig-6-1\u0022 class=\u0022xref-fig\u0022 href=\u0022#F6\u0022\u003E6\u003C\/a\u003E\u003Cem\u003EA\u003C\/em\u003E). Subtracting currents for the control from those during 5-HT application yielded 5-HT-induced currents at various membrane potentials. As illustrated in Figure \u003Ca id=\u0022xref-fig-6-2\u0022 class=\u0022xref-fig\u0022 href=\u0022#F6\u0022\u003E6\u003C\/a\u003E\u003Cem\u003EB\u003C\/em\u003E, the currents decreased with hyperpolarization but did not reverse at the membrane potential range examined (\u2212145 to \u221255 mV). Data can be fitted reliably by a linear regression line, and through extrapolation, the reversal potential was estimated to be \u2212145 mV. Similar results were obtained from eight other cells, and the mean estimated reversal potential was \u2212148 \u00b1 3 mV (\u003Cem\u003En\u003C\/em\u003E = 9). This value is far from the equilibrium potentials calculated for K\u003Csup\u003E+\u003C\/sup\u003E(\u003Cem\u003EE\u003C\/em\u003E\u003Csub\u003EK\u003C\/sub\u003E, \u221295 mV) or Cl\u003Csup\u003E\u2212\u003C\/sup\u003E (\u221254 mV).\n\u003C\/p\u003E\u003Cdiv id=\u0022F6\u0022 class=\u0022fig pos-float odd\u0022\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F6.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022I\u0026#x2013;V relationship of 5-HT-induced whole-cell currents. A, Whole-cell currents in response to a voltage ramp (top trace) before (control, thin trace indicated by an arrow), during (5-HT, bold trace indicated by an arrow), and 10 min after (wash, thin trace, unlabeled) 5-HT application (10 \u0026#x3BC;m, 2 min) in a neuron at P10. Ramps were applied at 20 sec intervals, and each trace was the average of three to four consecutive ramp responses. TTX (0.3 \u0026#x3BC;m) was present throughout the recording. Data were filtered at 100 Hz and digitized at 250 Hz. The dashed line indicates 0 pA. B,I\u0026#x2013;V relationship obtained by subtracting the control currents from those during 5-HT application. Data were fitted by a linear regression line (R2 = 0.98). The estimated reversal potential was \u0026#x2212;145 mV as determined by extrapolation.Vm, Membrane potential.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-1200318417\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;em\u0026gt;I\u0026lt;\/em\u0026gt;\u0026#x2013;\u0026lt;em\u0026gt;V\u0026lt;\/em\u0026gt; relationship of 5-HT-induced whole-cell currents. \u0026lt;em\u0026gt;A\u0026lt;\/em\u0026gt;, Whole-cell currents in response to a voltage ramp (top trace) before (control, thin trace indicated by an arrow), during (5-HT, bold trace indicated by an arrow), and 10 min after (wash, thin trace, unlabeled) 5-HT application (10 \u0026#x3BC;m, 2 min) in a neuron at P10. Ramps were applied at 20 sec intervals, and each trace was the average of three to four consecutive ramp responses. TTX (0.3 \u0026#x3BC;m) was present throughout the recording. Data were filtered at 100 Hz and digitized at 250 Hz. The dashed line indicates 0 pA. \u0026lt;em\u0026gt;B\u0026lt;\/em\u0026gt;,\u0026lt;em\u0026gt;I\u0026lt;\/em\u0026gt;\u0026#x2013;\u0026lt;em\u0026gt;V\u0026lt;\/em\u0026gt; relationship obtained by subtracting the control currents from those during 5-HT application. Data were fitted by a linear regression line (\u0026lt;em\u0026gt;R\u0026lt;\/em\u0026gt;\u0026lt;sup\u0026gt;2\u0026lt;\/sup\u0026gt; = 0.98). The estimated reversal potential was \u0026#x2212;145 mV as determined by extrapolation.\u0026lt;em\u0026gt;V\u0026lt;\/em\u0026gt;\u0026lt;sub\u0026gt;m\u0026lt;\/sub\u0026gt;, Membrane potential.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 6.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F6.medium.gif\u0022 width=\u0022227\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 6.\u0022 src=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F6.medium.gif\u0022 width=\u0022227\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F6.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 6.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/23\/8\/3373\/F6.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/452159\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 6.\u003C\/span\u003E \u003Cp id=\u0022p-30\u0022 class=\u0022first-child\u0022\u003E\u003Cem\u003EI\u003C\/em\u003E\u2013\u003Cem\u003EV\u003C\/em\u003E relationship of 5-HT-induced whole-cell currents. \u003Cem\u003EA\u003C\/em\u003E, Whole-cell currents in response to a voltage ramp (top trace) before (control, thin trace indicated by an arrow), during (5-HT, bold trace indicated by an arrow), and 10 min after (wash, thin trace, unlabeled) 5-HT application (10 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E, 2 min) in a neuron at P10. Ramps were applied at 20 sec intervals, and each trace was the average of three to four consecutive ramp responses. TTX (0.3 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) was present throughout the recording. Data were filtered at 100 Hz and digitized at 250 Hz. The dashed line indicates 0 pA. \u003Cem\u003EB\u003C\/em\u003E,\u003Cem\u003EI\u003C\/em\u003E\u2013\u003Cem\u003EV\u003C\/em\u003E relationship obtained by subtracting the control currents from those during 5-HT application. Data were fitted by a linear regression line (\u003Cem\u003ER\u003C\/em\u003E\u003Csup\u003E2\u003C\/sup\u003E = 0.98). The estimated reversal potential was \u2212145 mV as determined by extrapolation.\u003Cem\u003EV\u003C\/em\u003E\u003Csub\u003Em\u003C\/sub\u003E, Membrane potential.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cp id=\u0022p-31\u0022\u003ETaking into account the lack of effect by 5-HT on input resistance, one possibility is that 5-HT produces two different changes in membrane conductance: (1) a reduction of outward currents that are normally open at resting potentials (RPs), therefore likely of K\u003Csup\u003E+\u003C\/sup\u003E, and (2) an activation of inward currents that are carried by Na\u003Csup\u003E+\u003C\/sup\u003E, presumably via cation nonspecific channels that have a reversal potential near 0 mV. This hypothesis clearly explains the effects (or the lack of) of 5-HT on input resistance because the two actions of 5-HT produce opposite changes in input resistance, therefore canceling each other. This hypothesis is also consistent with the fact that 5-HT-induced currents did not reverse at membrane potential between\u003Cem\u003EE\u003C\/em\u003E\u003Csub\u003Ek\u003C\/sub\u003E and 0 mV (Fig.\u003Ca id=\u0022xref-fig-6-3\u0022 class=\u0022xref-fig\u0022 href=\u0022#F6\u0022\u003E6\u003C\/a\u003E\u003Cem\u003EB\u003C\/em\u003E), as explained mathematically in an elegant study (\u003Ca id=\u0022xref-ref-10-1\u0022 class=\u0022xref-bibr\u0022 href=\u0022#ref-10\u0022\u003EBrown et al., 1971\u003C\/a\u003E).\u003C\/p\u003E\u003Cp id=\u0022p-32\u0022\u003EThis hypothesis was examined in several experiments. First, 5-HT-induced responses were examined in ACSF containing high K\u003Csup\u003E+\u003C\/sup\u003E (7 m\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E instead of 3 m\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E), and the \u003Cem\u003EI\u003C\/em\u003E\u2013\u003Cem\u003EV\u003C\/em\u003E relationship was examined in the presence of TTX (0.3 \u03bc\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003E) with a ramp protocol (Fig. \u003Ca id=\u0022xref-fig-7-1\u0022 class=\u0022xref-fig\u0022 href=\u0022#F7\u0022\u003E7\u003C\/a\u003E\u003Cem\u003EA\u003C\/em\u003E). As shown in Figure \u003Ca id=\u0022xref-fig-7-2\u0022 class=\u0022xref-fig\u0022 href=\u0022#F7\u0022\u003E7\u003C\/a\u003E\u003Cem\u003EB\u003C\/em\u003E, the \u003Cem\u003EI\u003C\/em\u003E\u2013\u003Cem\u003EV\u003C\/em\u003E curve in high K\u003Csup\u003E+\u003C\/sup\u003E can be fitted reliably by a linear regression line with a reversal potential of \u2212112 mV. Similar responses were obtained from four cells, and the mean reversal potential was \u2212125 \u00b1 2 mV. Compared with the estimated reversal potential in normal ASCF (3 m\u003Cspan class=\u0022sc\u0022\u003Em\u003C\/span\u003EK\u003Csup\u003E+\u003C\/sup\u003E) (Fig. \u003Ca id=\u0022xref-fig-6-4\u0022 class=\u0022xref-fig\u0022 href=\u0022#F6\u0022\u003E6\u003C\/a\u003E\u003Cem\u003EB\u003C\/em\u003E), this is a shift of 23 mV, close to the predicted shift of 21 mV for\u003Cem\u003EE\u003C\/em\u003E\u003Csub\u003Ek\u003C\/sub\u003E. This result suggests that K\u003Csup\u003E+\u003C\/sup\u003E conductance is involved in 5-HT-induced responses.\n\u003C\/p\u003E\u003Cdiv id=\u0022F7\u0022 class=\u0022fig pos-float odd\u0022\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.jneurosci.org\/content\/jneuro\/	https://www.jneurosci.org/panels_ajax_tab/jnl_sfneneuro_tab_art/node:361787/1
Processes \| Free Full-Text \| Research on Fault Diagnosis of PST Electro-Hydraulic Control System of Heavy Tractor Based on Support Vector Machine Due to the harsh working environment of the tractor, the transmission can often be faulty. In order to ensure the reliability of its operation, it must be monitored and the fault discovered. In this paper, the support vector machine (SVM) method is used. The eigenvector conversion of the original data uses the following eigenvectors: Three fault modes (leakage fault of shift clutch hydraulic cylinder, blockage fault of oil passage, and blockage fault of proportional valve spool) are identified in matrix and laboratory (MATLAB) with the help of the library for support vector machines (LibSVM) toolkit, and the classification accuracy of test samples is 90%. The normal mode of the PST electro-hydraulic system and the three kinds of fault modes mentioned above are discriminated against, and the correct rate of fault diagnosis reaches 95%, which meets the needs of practical engineering. Analysis of the fault recording data of the power shifting transmission shift solenoid valve shows that the difference between fault pressure data and normal data is small, and the value of traffic data is greater. This method can realize the fault mode online recognition based on controller area network (CAN) communication, and the research results provide a theoretical basis for the fault diagnosis of the PST electro-hydraulic control system. Research on Fault Diagnosis of PST Electro-Hydraulic Control System of Heavy Tractor Based on Support Vector Machine by Huiting Ni , Liqun Lu * , Meng Sun , Xin Bai and Yongfang Yin Author to whom correspondence should be addressed. Processes 2022 , 10 (4), 791; https://doi.org/10.3390/pr10040791 Received: 14 March 2022 / Revised: 12 April 2022 / Accepted: 14 April 2022 / Published: 18 April 2022 (This article belongs to the Special Issue Application of Fuzzy Control in Computational Intelligence ) Abstract : Due to the harsh working environment of the tractor, the transmission can often be faulty. In order to ensure the reliability of its operation, it must be monitored and the fault discovered. In this paper, the support vector machine (SVM) method is used. The eigenvector conversion of the original data uses the following eigenvectors: Three fault modes (leakage fault of shift clutch hydraulic cylinder, blockage fault of oil passage, and blockage fault of proportional valve spool) are identified in matrix and laboratory (MATLAB) with the help of the library for support vector machines (LibSVM) toolkit, and the classification accuracy of test samples is 90%. The normal mode of the PST electro-hydraulic system and the three kinds of fault modes mentioned above are discriminated against, and the correct rate of fault diagnosis reaches 95%, which meets the needs of practical engineering. Analysis of the fault recording data of the power shifting transmission shift solenoid valve shows that the difference between fault pressure data and normal data is small, and the value of traffic data is greater. This method can realize the fault mode online recognition based on controller area network (CAN) communication, and the research results provide a theoretical basis for the fault diagnosis of the PST electro-hydraulic control system. Keywords: component ; tractor power-shift transmission (PST) ; controller area network (CAN) bus ; support vector method (SVM) ; troubleshooting 1. Introduction In this paper, a tractor with full power shift transmission developed independently is seen as the research object. At present, there is little research on the diagnosis of faults in the electro-hydraulic system of power shift transmission, so it is necessary to consider and explore this field. The application layer of the CAN bus contains the data frame of the shift solenoid valve parameters. The fault diagnosis: The Electronic Control Unit (ECU) obtains real data through CAN network data sharing, monitors the status of transmission, and diagnoses the faults of electro-hydraulic systems through data analysis and operation, as well as analyzing the characteristics of each fault by simulating the flow or clutch pressure response of the proportional valve in fault mode, screening and processing previous fault record data, utilizing the support vector machine (SVM) classification method, and utilizing the library for support vector machines (LibSVM) toolkit to realize real-time fault pattern recognition in matrix and laboratory (MATLAB). 2. Failure Analysis of PST Electro-Hydraulic System 2.1. How a Full Power Shift Transmission Works PST is an important transmission device, which is mainly used for heavy-duty vehicles and hydraulically driven wheeled construction machinery or agricultural machinery. Its basic structure is a hydraulic-mechanical system without a main clutch. The shift actuator is a hydraulically driven multi-plate wet clutch. The shift operation can be achieved through the shift solenoid valve. The power transmission is marked by no interruption of power, fast shift speed, and no impact. In this paper, a fixed-shaft multi-shaft tandem structure power shift transmission is designed, including power shift and power shift parts; all gears can realize the power shift, so it is called a “full power shift transmission.” Figure 1 shows the internal structure of the full power shift transmission designed in this paper, which consists of a main transmission device and an auxiliary transmission device, which are connected in parallel. Electro-Hydraulic Proportional Control System Figure 2 shows the electro-hydraulic control system of the PST. The single-chip microcomputer is the control part, and the hydraulic module is the execution part. The hydraulic module is split into a pressure regulation module and a cooling and lubrication module (L1, L2, L3). In the power shift transmission designed in this paper, the most critical is the shift proportional solenoid valve. The structure of the shift proportional solenoid valve is shown in Figure 3 . The shift proportional solenoid valve is composed of an adjusting screw, an adjusting spring, a valve sleeve, a coil, an armature, a valve body, a valve baffle, a valve core, and an oil inlet ball valve. It works on the principle that the position of the adjustment screw determines the preset pressure. The coil is energized to generate a magnetic field, attracting the armature to the approach. The ball valve moves to the left, and pressure oil fills the clutch oil circuit. The magnitude of the current determines the strength of the magnetic field and the degree to which the oil inlet is opened. By adjusting the duty ratio of the Pulse Width Modulation (PWM) signal to control the size of the opening current, the continuous adjustment of the pressure and flow is realized, and the soft engagement of the shift clutch is controlled. The valve port of the shift proportional solenoid valve is generally a cone valve or a ball valve structure, and an annular gap will be formed between the valve core and the valve seat during the opening and closing process, resulting in a throttling effect. The flow characteristics through this gap are: Q c = C v A v 2 Δ p v ρ (1) In the formula, C v is the flow coefficient A v is the flow area of the valve port, m 2 ; Δ p v is the pressure difference between the inlet and outlet of the solenoid valve, Δ p v = p s − p d , Pa; p s and p d are the inlet and outlet pressures of the proportional solenoid valve, respectively; and ρ is the oil density, kg/m 3 . The flow coefficient is usually related to the Reynolds coefficient flowing through the gap of the valve port, as well as the geometry and size of the valve port, etc. It is a very complex dimensionless coefficient. The flow coefficient under a certain condition is usually obtained by experiments, and the general value range is 0.60–0.80. The oil pressure p c of the shift clutch cylinder can be expressed as: p c = p s − K p 2 Q 2 (2) In the formula, p s is the system oil pressure, about 2 MPa. At present, the shift proportional solenoid valve of the pressure regulation module mostly adopts an open-loop control method without signal feedback. For example, the shift control signal of a power shift transmission of Lovol Apos is illustrated in Figure 4 . The electrical signal range is 340–1100 mA. After being supplemented by the amplifier, it sends a DC current to the proportional electromagnet of the electro-hydraulic proportional valve. The proportional electromagnetic force is generated in proportion to pushing the spool to produce the corresponding displacement, thereby controlling the flow, pressure, and direction of the hydraulic oil entering the multi-plate wet clutch and the clutch friction plates from slipping to engaging, so that the load obtains the corresponding force, displacement, and speed. The accumulator can buffer pressure in the system and play the role of regulating flow and pressure. 2.2. Electronic Control System Failure Electronic control system faults are divided into sensor faults, electronic control unit faults, electronic control element circuits, and CAN communication faults, as shown in Table 1 . TCU control of the power gear in the automatic mode. The more gears, the greater the control difficulty. The main input and output signals of the TCU are presented in Figure 5 . The input signals of the PST control unit mainly include the driver’s driving intention signal, the engine speed torque signal, the shift actuator feedback signal, the final drive, and the walking system signal, etc. The PST- Electronic Control Unit (ECU) needs to calculate a reasonable shift control law according to a certain judgment logic or algorithm for the above signals and realize the control of the shift actuator—the shift solenoid valve. The shifting process is illustrated in Figure 6 . The electronic control network sends messages through the CAN bus. The communication module chooses to accept the relevant messages from the CAN bus and converts them into the data required for the controller to compute. After the data processing module processes and converts some of the signals, it is sent to the shift decision module, and the control current of the shift solenoid valve is formed after the calculation of multiple signals, which indirectly control the hydraulic oil pressure and complete a power shift. Each signal is produced by its corresponding sensor. Once the sensor fails, it will influence the decision-making of the controller. Types of sensor faults can be divided into voltage-based faults and time-based faults. The probability of the intramural program’s runaway failure of the electronic control unit is small. Once it occurs, the TCU will no longer communicate with the VCU, and the VCU will respond according to a reliable fault tolerance strategy to ensure the safety of operation or driving. CAN communication failure is manifested as the failure of the VCU to receive the TCU signal and the loss of some TCU signals. When the pressure reducing valve or the gear solenoid valve fails, the gear selection and shifting disorder occurs. That is, the shifting clutch cannot execute the selection and shifting commands of the TCU. For the fault diagnosis of the shifting mechanism, the actual gear and the gear sensor displayed by the speed ratio of the input shaft and the output shaft are used to display the gear, and the TCU needs the gear to analyze the redundancy between the three so as to accurately diagnose the fault condition [ 10 ]. 2.3. PST Hydraulic System Failure Due to the sudden change of direction of the shift solenoid valve and the engagement or separation of the shift clutch, a pressure shock will be generated in the electro-hydraulic system, causing the system pressure to fluctuate rapidly in a short period of time, easily causing damage to the control elements and sealing devices and causing vibration and noise. Electro-hydraulic system failures include pump-motor system failure, proportional reversing valve failure, shift clutch failure, etc., as well as any component of the hydraulic system such as the charge pump, cooler, and pipeline related to it; common transmission failures and failure caused by the maintenance method are shown in Table 2 . The traditional fault diagnosis method is manual inspection, and the cause of the fault and the fault location are determined and repaired through the methods of mechanical inspection and hydraulic inspection. Among them, hydraulic detection looks for faults in three aspects: the oil level of the transmission oil pans, the oil leakage of the shifting clutch, and the cooling and lubricating oil amount of the torque converter. For example, if the oil level in the transmission oil pan is low, the clutch operating pressure will be low and the output flow of the torque converter charge pump will be insufficient. The oil level of the transmission oil pan is monitored by installing a liquid level sensor, and the liquid level data are displayed on the instrument. With an alarm when the oil level is low, such failure can be avoided. When the seal ring of the clutch cylinder or the seal connected to it is damaged, the system will have a large leakage. The leakage and the degree of damage can be judged by the data measured by the flow sensor of the clutch oil inlet pipeline. The flow simulation curve of the clutch leakage failure mode is shown in Figure 7 a, and the area difference between the green dotted line and the red solid line represents the leakage amount. Under normal conditions, the leakage of all shift clutches is basically the same. If the leakage of one of the clutches exceeds the standard, the clutch should be checked and repaired immediately. Figure 7 b shows the flow change curve of the clutch oil filling process in blocking mode. The response of the whole process lags, and the maximum value is always lower than the maximum value in the normal state. The more severe the lag and the smaller the flow, the more severe the blockage. 3. Fault Diagnosis of PST Electro-Liquid System Based on CAN Bus Network 3.1. Fault Diagnosis Principle The traditional fault diagnosis method is manual inspection, which relies on skilled engineers and experts to deal with the problems that affect the efficiency of tractor operation. The fault diagnosis of the tractor PST electro-hydraulic system based on the CAN network relies on the analysis of various sensors, the fault diagnosis ECU, and the prompt and alarm of the instrument/virtual terminal to complete the fault diagnosis and obtain maintenance. Based on the CAN bus, the principle of fault diagnosis, as shown in Figure 8 , the fault diagnosis process requires the equipment bus, the tractor bus, and multiple modules, and the information acquisition module collects the flow of each shift solenoid valve. Through the VCU gateway completion information in the network, the fault diagnosis of the ECU determines the fault point and the fault type, then displays the exact fault information through the meter/virtual terminal or alert to complete the task of troubleshooting. At present, commonly used intelligent algorithms are Fuzzy Logic Algorithms (FLA), Neural Network Algorithms (NNA), SVM, and so on. The FLA depends on the establishment of a knowledge base, the performance of the inference engine, and expert experience. The NNA needs a lot of sample data for its training network, and it is not difficult to fall into a local optimum. The test sample data for this experiment have the characteristics of a small sample size and high dimensionality. SVM has a good classification and generalization ability when there are very few samples and has superior ability in classification and prediction. For high-dimensional nonlinear problems, SVM can be transformed into linear problems by constructing linear discriminant functions in high-dimensional space, so the complexity of the SVM algorithm will not have anything to do with the dimension of vectors. Moreover, the algorithm will be transformed into a convex quadratic programming problem with linear constraints. In theory, the global optimal solution can still be found, and the local extremum problem in NNA and other methods can be addressed. In view of the limited number of samples obtained in this paper, it has been decided to adopt the support vector machine algorithm to identify several failure modes online and use the support vector machine as the core algorithm of the fault diagnosis module of ECU power shift fault diagnosis. 3.2. SVM Principle SVM is a new learning method in statistical learning theory that adopts the principle of structural risk minimization [ 10 ]. It shows many advantages in pattern recognition or the classification of small samples, as well as nonlinear and high-dimensional data samples, and has a strong generalization ability, which can realize linear and nonlinear classification problems. 3.2.1. Linear Scalability The basic idea of SVM can be explained using the example of two-dimensional data samples in Figure 9 . Black spots and white spots are two different samples, and in the case of linearity, the sample is set to (x i , y i ), i = 1,2,…, m, x i ∈ Rd, y i ∈ {+1,−1} and the linear discrimination function is f(x) = ω x + b , that is, the classification plane is: ωx + b = 0 (3) Figure 9. Optimal classification surface in two-dimensional. Figure 9. Optimal classification surface in two-dimensional. The two-dimensional extension of the multidimensional = (ω 1 , ω 2 , ω n ) classified super plane’s space is as follows: ω T x + b = 0 (4) Two types of samples in multidimensional space can be expressed as: ω T · x + b ≥ + 1 ω T · x + b ≤ − 1 (5) There are also some data points distributed on the super plane ω T x + b = ±1, called the support vector, it is also a support vector to separate the data points in the multidimensional space. According to the geometric relationship, the distance from the support vector to the optimal plane is 1 / ‖ ω ∥ , and the classification interval is 2 / ‖ ω ∥ . Make the classification interval to obtain the optimal plane. The constraints are located on both sides of the super plane to obtain an inequality constraint optimization problem: max 2 ‖ ω ∥ s . t . y i ω T x i + b ≥ 1 , i = 1 , 2 , ⋯ , (6) For convenience of calculation, the transformation of the above formula into the minimization problem proceeds as follows: min 1 2 ‖ ω ∥ 2 s . t . y i ω T x i + b ≥ i = 1 , 2 , ⋯ , m (7) Equation (7) is a convex quadratic programming problem. In order to further simplify the solution, it is transformed into a Lagrange dual problem, and the corresponding Lagrange multiplier α i > 0 is set for the inequality constraints of Equation (7). Obtain the Lagrange function ω , b of the original problem: L ω , α = 1 2 ‖ ω ∥ 2 + ∑ i = 1 m α i [ 1 − y i x i + b (8) The optimization goal is: max α i min ω , b L ω , b , α , for the Lagrange function to seek its minimum and questions about α i , just a deflection is zero, then connect Equation (8) to get ω, b, α i : max α ∑ i = 1 m α i − 1 2 ∑ i = 1 m ∑ j = 1 m α i α j y i y j x i T x j s . t . ∑ i = 1 m α i y i = 0 α i ≥ 0 (9) As seen from the Equation (9), by minimizing ω,b so that it is small, the optimization function is only the α parameter. This problem is to solve the sequential minimal optimization (SMO) algorithm [ 11 ], solving the α * value of α . If α* is the optimal solution, the above-described dual problem has a unique solution ( ω , b ), and it is: ω ∗ = ∑ i = 1 m α i ∗ y i x i (10) In the Equation (10), the α i ∗ non-zero corresponding sample is a support vector, so the power coefficient vector of the optimal classification surface is a linear combination of support vectors, and b * is the classified threshold, so it can be solved by constraint conditions α i [y i ( ω i x i + b ) − 1] = 0. Solving the above problems, the optimal classification surface function is: f x = s g n ω T x + b = s g n [ ∑ i = 1 m α i ∗ y i x i · x + b ∗ ] (11) 3.2.2. Linear Indiscriminate Problem At data acquisition, there is noise data, so that the original simple classification becomes linear. The SVM introduces the loan tolerance mechanism of the slack variable and the penalty factor, forming the algorithm itself, so that the distance from the classification plane does not narrow, and the soft interval is realized. In this classification, linear inseparable problems are described as: min ω , b , ξ 1 2 ‖ ω ∥ 2 + C ∑ i = 1 m ξ i s . t . y i ω · φ x i + b ≥ 1 − ξ i ξ 0 , i = 1 , 2 , ⋯ , m (12) In the Equation (12), ξ is a relaxation variable, indicating that the data point is far from the classification plane, and C is a penalty factor, which controls the degree of penalty of the missed data point. The Lagrange function is as follows: L ω , b , β = 1 2 ‖ ω ∥ 2 + C ∑ i = 1 m ξ i − ∑ i m α i y i ω · x i + b − 1 + ξ i − ∑ i = 1 i ξ i (13) In Equation (13), α i and β i are non-negative Lagrange multipliers, and an optimization problem can be converted to the occasional problem by seeking ω , b and ξ: max α ∑ i = 1 m α i − 1 2 ∑ i = 1 m ∑ j = 1 m α i α j y i y j K x i , x j s . t . ∑ n = 1 m α i y i = 0 0 ≤ α i ≤ C , i = 1 , 2 , ⋯ m (14) Several cases of solving α i are: ① α i = 0; ② 0 < α i < C ; ③ α i = C . ②, ③ correspond to the support vector x i , where x i is called ② standard support vector, and x i in ③ is called a boundary support vector. If the solution to the optimization problem is α * = ( α * 1 , α * 2 , L, α n ) T : ω ∗ = ∑ i = 1 m α i ∗ y i x i b ∗ = y j − ∑ i = 1 m α i ∗ y i x i · x j (15) By solving the above problems, ultimately transforming linearized inseparable problems into linear can be linear, and the optimal classification surface function can be obtained. 3.2.3. Low-Dimensional Space Nonlinear Problem When the data set is inadequate in the low-dimensional space, the data set is mapped to the high-dimensional space through the core function. If the data set in the high dimension is linear, the super plane is expressed as: ω T φ ( x ) + b = 0 Derived from the form of Equation (16) max α ∑ i = 1 m α i − ∑ i = 1 m ∑ j = 1 m α i α j y i y φ x i T φ x j s . t . ∑ i = 1 m α i y i = 0 ≥ 0 (17) φ(x i ) T φ(x j ) T in Equation (17) is regarded as a whole, and a function of x i and x j , namely κ ( x i , x j ) = φ ( x i ) T φ ( x j ) T ; κ ( x i , x j ) is called the kernel function. Commonly used kernel functions include linear kernel functions, polynomial kernel functions, Gaussian kernel functions, and Sigmoid kernel functions. In fact, the kernel function has no fixed form, and can be understood as a tool for mapping data sets from low-dimensional space to high-dimensional space. Figure 10 vividly shows the mapping function of the Gaussian kernel function. Experience shows that the Gaussian kernel function has the best mapping effect. To sum up, the application of SVM in classification can be divided into two situations: (1) a linear classification problem; (2) a nonlinear classification problem. The problem (1) can be divided into two cases: ① When the problem is linearly separable, only the classified linear function (two-dimensional space) under the linear constraint condition is needed, and the structural risk is 0. ② When the problem is linearly indivisible, penalty factor C and relaxation variable ξ should be introduced, where ξ is the degree of “tolerance” to the wrong sample, C is the degree of punishment for this kind of “tolerance”, the soft interval realizes the sample separability, and the structural risk is not higher than C. In problem (2), the linear inseparability of low-dimensional space is mapped to high-dimensional space by the kernel function, thus realizing linear separability. 3.2.4. Solution of SVM in Multi-Class Classification Problem At present, the multi-class promotion of SVM mainly adopts two methods: multi-objective optimization and combination coding [ 12 ]. The multi-objective optimization method solves all classification problems at once, and combination coding is realized by constructing multiple two-class SVMs with the following specific methods [ 13 ], as shown in Table 3 . 4. An Example of Fault Classification Prediction and Result Analysis 4.1. Fault Case Analysis According to the data recorded in the past, the faults of the hydraulic subsystem of the shift clutch mainly include leakage faults caused by poor sealing, oil passage blockage faults, and valve core jam faults. 4.1.1. Normal Mode When the shift clutch works normally, the oil pressure, flow rate and other indicators are normal, the shift clutch moves quickly, the combination is smooth, there is no power interruption and impact in the shift process, and the pressure is constant. 4.1.2. Sealing Ring Damage and Leakage Fault With frequent use or improper operation, the sealing ring inside the wet shift clutch is easily affected by friction heat, which causes deformation and damage, resulting in clutch oil leakage fault. 4.1.3. Oil Passage Blockage Fault The hydraulic system has to be polluted by dust and other solid particles in the air, so the phenomenon of oil passage blockage is very common. In the electro-hydraulic system for proportional control of the shifting clutch studied in this paper, the outlet joint between the solenoid valve plate and the valve core is most prone to blockage. The accumulation of impurities reduces the diameter of the pipeline, decreases the flow rate in and out of the clutch cylinder, and increases the time of oil charging and discharging. The increase in oil charging and discharging will lead to more friction heat when the shift clutch goes hand in hand with power, which will aggravate the wear and even burn off the clutch. 4.1.4. Proportional Valve Spool Jam Fault When the return spring of the proportional valve fails, it cannot return normally, and the oil cylinder of the shift clutch keeps the engagement pressure all the time, which leads to a large number of sliding wear losses caused by the friction plate being in a sliding wear state for a long time, and the generated heat will cause the friction plate to burn down, which is a very serious fault. By analyzing the fault record data of the shift solenoid valve of power shift transmission, it is found that the difference between fault pressure data and normal data is small, and the increment of flow data fluctuates greatly. The following eigenvectors are used to transform the original data: root mean square value X RMS , peak factor C , kurtosis factor K , pulse factor I , and waveform factor S [ 14 ], which are defined as follows: X R M S = 1 N ∑ i = x i 2 (18) C = max x i X R M S (19) K = ∑ i = 1 N X i 4 N X R M S 4 (20) I = max x i 1 N ∑ i = 1 N x i (21) S = X R M S 1 N ∑ i = 1 N x i (22) Report to the general assembly on the flow rate. The difference between the pressure data and the normal data is poor. For the convenience of calculation and processing, only the root means square value X RMS-P is considered. The flow rate and pressure value of the first second after the change section are taken, and the sampling point interval of the data is 0.05 s. T, F1, F2, and F3 are used to represent normal mode, hydraulic cylinder leakage of the shift clutch, oil passage blockage, and valve core blockage of the proportional valve, respectively. Table 4 indicates the sample data of the eigenvector after fault data processing. 4.2. Application of LibSVM in MATLAB LibSVM software is a simple, fast, and effective software for SVM pattern recognition and regression. The software can solve classification problems such as C-SVC and v-SVC, one-class SVM distribution estimation problems, epsilon-SVR, and v-SVR regression problems. Four kernel function types are provided: linear kernel, polynomial kernel, radial basis function, and sigmoid kernel. In addition, the kernel function dimension d , the slack variable parameter γ of the kernel function, the penalty factor e , and other parameters is set, and the selected type only needs to be indicated when writing the program. LibSVM software can effectively solve multi-class classification problems, select parameters for cross-validation, weight unbalanced samples, and estimate the probability of multi-class problems [ 15 ]. In this paper, the software of this version is selected to be installed in Matlab2013a to realize the construction of a computer analysis environment. The number of training samples selected in this paper is 5. That is, training and predictive sample sets of each pattern are 5. This paper uses the default C-SVC classifier to classify and diagnose, and implements the following steps: Select “Matlab2013a\toolbox\libsvm3.23\windows” path in Matlab’s control window, establish a dataset “mat” file “truedatas.mat, faultdatas1.mat, faultdatas2.mat, faultdatas3.mat” and a category label set “lableset1.mat, lableset2.mat, lableset3.mat, lableset4.mat”. Making the training set and test set data, the call function mapminmax scaled the test set to the [0,1] section. Test Set Sample Training Requires calls Svmtrain function, model = svmtrain(training_label_vector, training_instance_matrix,’libsvm_options’);where libsvm_options options include SVM type, core function type, core function parameter selection. In this document model = svmtrain(train_set_labels, train_set, ‘-c 1 -g 0.07’);’-c 1 -g 0.07’ omitted the SVM type, the default is C-SVC, omitted the core function type, the default is the radial base RBF, the default value of the parameter C is 1, g default value 1/k (k is sample data) Dimension, this article k = 6), the fault tolerant factor is 0.07. Using the grid search method to find the optimal C and g combination, select the value setting for gamma in [0.001,0.01,0.1,1,10,100]: for C in [0.001,0.01,0.1,1,10,100]:, then call the command svm = SVC(gamma = gamma, C=C), conduct a training for each combination, and then find a large set of parameter values for the minimum error to find the value of g = 0.001, C = 100. This method is time consuming, but it can accurately find the best parameters. The prediction set is classified by the model described above, and the Svmpredict function, [predict_label] = svmpredict (test_set_labels, test_set, model); the predictive sample test_set is corresponding to the label set test_set_labels, and the classification correct rate can be calculated. 4.3. Test Results Analysis The outputs of T, F1, F2 and F3 are represented by numbers 0, 1, 2 and 3, respectively. The classification results of the prediction set in the sample set in Table 1 are shown in Figure 11 , which shows the classification results more intuitively. The results show that two samples of F1 failure mode are incorrectly predicted as normal mode, but all the samples of other failure modes are correctly predicted. Therefore, the classification accuracy of predicted samples is 90%. optimization finished, #iter = 5 nu = 1.000000 obj = −7.183243, rho = −0.025135 nSV = 10, nBSV = 10 Total nSV = 20 Accuracy = 90% (18/20) (classification) A Fisher criterion is introduced here to analyze the diagnosis error of F1 internal leakage fault, and its principle will not be described in detail [ 16 ]. If the intra-class dispersion value of a feature is smaller and the inter-class dispersion value is larger, the Fisher criterion value is higher. According to pairwise discrimination, the Fisher criterion for calculating the characteristic variables of F1-F3 and T modes is shown in Table 5 : F2 has a larger Fisher criterion value of 3.253002, and F3 has a larger Fisher criterion value of 1.369812 and 6.105690, so the accuracy rate is higher when predicting classification, and each Fisher criterion value of F1 is very small. The reason is that the occurrence of faults has a gradual change, which is difficult to detect in the initial stage of internal leakage. Only when the damage degree of shift clutch piston changes from quantitative change to qualitative change will the system be strongly affected. Due to the limited samples of the prediction set, this paper will use all 40 samples to further test the model, and the simulation results are shown in Figure 12 . optimization finished, #iter = 5 obj = −7.647503, rho = −0.015225 nSV = 10, nBSV = 10 Total nSV = 20 Accuracy = 95% (38/40) (classification) From the running results, misclassified samples are still two samples of F1 failure mode, and all other samples are correctly identified. The increase of the number of test samples further improves the prediction accuracy to 95%. 4.4. Factors Affecting the Correctness of Classification 4.4.1. Training Sample Quantity The literature was verified by simulation and concluded that the accuracy of leakage diagnosis of hydraulic systems would not increase with the increase of training samples, indicating that SVM has a significant advantage in solving the problem of pattern recognition of small samples [ 16 ]. Table 6 shows the relationship between the number of training samples and the correct rate. 4.4.2. Penalty Coefficient C and Radial Basis Function (RBF) Parameters γ Penalty coefficient C understands the width of the error. The greater the value of C, the more you can tolerate the error; if it is easier, the classification plane is too small, making the classification significant. The smaller the C value, the lower the classification accuracy; C is too large or too small, and the generalization ability of the model (adaptability to new samples) will deteriorate. γ is a parameter that comes with the RBF core, which determines the distribution of data mapping for new feature space. The larger the γ value, the less supported; the smaller the γ value, the more supported; the support vector affects the speed of training and forecasting. 5. Conclusions This paper analyzes the fault characteristics of the PST electro-hydraulic system and performs online fault pattern recognition based on the universal CAN bus. Since the number of faulty samples is limited, the support vector machine classification algorithm can better solve a number of small-sample, nonlinear, high-dimensional nonlinear problems, so the support vector machine is used to realize fault mode identification. The selection and calculation of the feature vector are made with the faulty data. The LibSVM toolkit is applied to the normal mode of the PST electro-hydraulic system and the three types of fault code in MATLAB. The result shows two samples of the F1 fault mode being erroneous. In order to return to normal mode, the samples of another fault mode are all predicted correctly, so the correct rate of the predictive sample is 90%. According to the Fisher criterion, misconceived samples are still two samples of F1 fault mode, and all other samples are correctly identified. With the increase of the number of test samples, the prediction accuracy is further improved to 95%, which meets the need of practical engineering. Author Contributions Conceptualization, H.N. and Y.Y.; methodology, H.N. and L.L.; software, H.N.; validation, H.N., M.S. and X.B.; formal analysis, H.N.; investigation, L.L.; resources, L.L.; data curation, X.B.; writing—original draft preparation, L.L.; writing—review and editing, L.L.; visualization, H.N.; supervision, X.B.; project administration, M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by RESEARCH AND DEVELOPMENT OF INTEGRATION CONTROL SYSTEM FOR INTELLIGENT HEAVY-DUTY TRACTORS, grant number 2016YFD070110102. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. References Ebrahimi, E.; Javadikia, P.; Astan, N.; Heydari, M.; Jalili, M.H.; Zarei, A. 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Full power shift transmission (partial) power transmission scheme. Figure 1. Full power shift transmission (partial) power transmission scheme. Figure 2. Principle of electro-hydraulic control of power shift transmission. Figure 2. Principle of electro-hydraulic control of power shift transmission. Figure 3. Structure diagram of shifting proportional solenoid valve. 1. Adjusting screw; 2. Adjusting spring; 3. Valve sleeve; 4. Coil; 5. Armature; 6. Valve body; 7. Valve baffle; 8. Spool; 9. Oil inlet ball valve. Figure 3. Structure diagram of shifting proportional solenoid valve. 1. Adjusting screw; 2. Adjusting spring; 3. Valve sleeve; 4. Coil; 5. Armature; 6. Valve body; 7. Valve baffle; 8. Spool; 9. Oil inlet ball valve. Figure 4. Input current signal of proportional electromagnet. Figure 4. Input current signal of proportional electromagnet. Figure 5. PST control principle. Figure 5. PST control principle. Figure 6. Power shift process. Figure 6. Power shift process. Figure 7. Flow characteristics of faults mode. ( a ) Flow characteristics of leak mode. ( b ) Flow characteristics of blockage mode. Figure 7. Flow characteristics of faults mode. ( a ) Flow characteristics of leak mode. ( b ) Flow characteristics of blockage mode. Figure 8. Block diagram of fault diagnosis of PST electro-hydraulic system based on CAN bus. Figure 8. Block diagram of fault diagnosis of PST electro-hydraulic system based on CAN bus. Figure 10. Mapping function of Gaussian kernel function. Figure 10. Mapping function of Gaussian kernel function. Figure 11. Test sample data classification results. Figure 11. Test sample data classification results. Figure 12. All sample prediction classification results. Figure 12. All sample prediction classification results. Table 1. TCU electronic control system fault. Table 1. TCU electronic control system fault. Fault Category Fault Specific Name Sensor Input shaft speed, output shaft speed sensor, shift clutch position sensor, gear position sensor, oil source pressure sensor, etc. Electronic control unit Program run away Electronic control element circuit Solenoid valve failure, solenoid valve drive circuit failure, power failure, etc. CAN communication The Vehicle Controller Unit (VCU) cannot receive the TCU signal, a certain signal of the TCU is lost, the CAN bus is short-circuited to the ground/power supply, or the circuit is disconnected. Table 2. Power shift wet clutch common fault and maintenance. Table 2. Power shift wet clutch common fault and maintenance. Common Error Cause of Issue Maintenance Method Low system pressure, normal clutch leakage 1. The oil level in the transmission oil pan is too low. 2. The spring of the pressure-regulating valve of the transmission control system is broken. 3. The spool of the pressure regulating valve is stuck. 4. The oil charge pump is working abnormally. 1. Pour in the appropriate amount of oil. 2. Replace the spring on the pressure regulating valve. 3. Clean the spool and sleeve of the pressure regulating valve. 4. Examine and repair the charge pump. Low system pressure and excessive clutch leakage 1. The piston seal of the clutch cylinder is damaged or severely worn. 2. The oil seal at the connection between the clutch outer drum and the shaft is worn or damaged. 3. The oil pump works abnormally. 1. Changing the sealing ring. 2. Changing the oil seal. 3. Replace or repair. Transmission overheating 1. The oil seal in the torque converter is worn, and the oil drain is too large. 2. The oil pump is worn. 3. The oil level in the transmission oil pan is too low. 4. The oil pump inlet line enters the air. 1. Replacing the oil seal. 2. Oil pump replacement. 3. Increasing the quantity of oil. 4. Inspect the oil inlet pipe joint and tighten it. Transmission is noisy 1. The pump wheel is severely worn. 2. The oil pump is worn. 3. The bearing is severely worn or damaged. 1. Changing the pump wheel. 2. Changing the oil pump. 3. Inspect the new bearing and replace it if necessary. Low flow through cooler and low torque converter inlet pressure 1. Torque converter safety valve spring is damaged. 2. A portion of the torque converter safety valve is opened. 3. The leakage in the torque converter is large. 4. The shift clutches oil seal is worn or damaged. 1. Change the spring on the torque converter safety valve. 2. Examining the torque converter safety valve ball seat for wearing. 3. Inspect torque converter assembly parts and replace any that are severely worn. 4. Install a new clutch oil seal. The flow through the cooler is small and the torque converter inlet pressure is high 1. If the pressure of the gearbox lubricating oil is low, the cooler is blocked. 2. The oil return line of the cooler is partially blocked. 3. If the pressure of the gearbox lubricating oil is too high, the lubricating oil circuit is blocked. 1. Vacuum the oil cooler. 2. Disinfect the oil return line. 3. Inspect transmission lubricating oil circuit and clear any clogged components. Table 3. Combination encoding method. Table 3. Combination encoding method. Classify Distinguish One against rest (OAR) OAR is the earliest and most frequently used method at present. Its basic principle is to construct k second-class classifiers (k is the number of classes) by taking a certain class of samples as one class and other classes as another. Samples have to be discriminated against by k classifiers and output k classification values. The determination of this method is that the number of training samples is large, and the calculation is complicated. One against one (OAO) The basic idea behind OAO is to create one SVM classifier for two types of samples at the same time, so k (k − 1)/2 classifiers are required for the classification of k samples. When testing, each of the two types is compared, and the calculation is complicated. In addition, there is a problem that the identical sample belongs to multiple types, and the classification accuracy is low. Directed Acyclic Graph (DAG) DAG is part of the extension of OAO. The distinction is located between the prediction stage, which requires only k − 1 discriminant functions for each test, and the execution stage, which requires k − 1 discriminant functions for each test. It is fast in calculation and avoids the disadvantage of inseparable samples. Binary Tree Multi-class Classification Algorithm Combining the binary decision tree with SVM, the multi-class samples are divided into two categories from the root node, and the two sub-nodes continue to divide their samples into two categories, and then go down until each sub-node contains one category. Each node is classified by a support vector machine classifier, which has the advantage of fast classification speed, but after a sample is disqualified at a certain node, the error will continue irreversibly. Direct classification algorithm The advantage of solving multi-class samples as a whole is that the number of constructed classifiers is small, but this method has no unified construction method, the training process is slow, and the classifier structure is complex. Table 4. Sample data. Table 4. Sample data. Number X C K I S XP Fault Type 1 3.718150 1.632139 1.590318 2.580348 1.580960 13.51565 T 2 3.718159 1.632138 1.590307 2.579709 1.580570 12.59522 T 3 3.718169 1.632137 1.590282 2.578267 1.579688 13.51165 T 4 3.718181 1.632131 1.590258 2.576918 1.578867 13.50904 T 5 3.718202 1.632125 1.590208 2.574224 1.577222 13.50390 T 6 3.71822 1.632117 1.590165 2.571917 1.575816 13.49945 T 7 3.718262 1.632101 1.590082 2.567598 1.573186 13.49108 T 8 3.718278 1.632094 1.590050 2.565904 1.572154 13.48779 T 9 3.718323 1.632077 1.589977 2.562131 1.569859 13.48042 T 10 3.718379 1.632053 1.589894 2.557801 1.567229 13.47198 T 11 4.641408 1.308204 1.577676 1.361743 1.040926 8.232270 F1 12 4.503738 1.348064 1.606603 1.425565 1.057490 8.814577 F1 13 4.374942 1.387634 1.627415 1.497733 1.079342 9.403138 F1 14 4.119849 1.473445 1.546716 1.635828 1.110206 10.30141 F1 15 3.986659 1.522576 1.552947 1.756648 1.153734 10.73721 F1 16 3.897091 1.557487 1.543815 1.870622 1.201051 11.49526 F1 17 3.829932 1.584728 1.571876 1.988583 1.254842 11.92903 F1 18 3.783049 1.604309 1.577237 2.106763 1.313190 12.33731 F1 19 3.752768 1.617206 1.581351 2.219805 1.372617 12.67724 F1 20 3.734874 1.624917 1.584432 2.322164 1.429097 12.94919 F1 21 1.909045 1.032249 1.508721 1.032450 1.000194 2.479529 F2 22 2.488536 1.094736 1.505060 1.154214 1.054331 6.068260 F2 23 2.939103 1.285464 1.513321 1.594527 1.240429 10.45648 F2 24 2.986767 1.456093 1.506423 2.011038 1.381119 11.75795 F2 25 3.141969 1.459963 1.515589 2.058338 1.409856 12.32977 F2 26 3.287192 1.513136 1.515163 2.162645 1.429247 12.45779 F2 27 3.416444 1.539293 1.512336 2.294414 1.490563 12.93440 F2 28 3.546686 1.540993 1.502323 2.297025 1.490613 12.94182 F2 29 3.447888 1.628382 1.518066 2.557372 1.570499 13.39684 F2 30 3.512039 1.629298 1.516488 2.575288 1.580612 13.47951 F2 31 3.068556 1.992056 1.775022 1.230561 1.802556 13.50322 F3 32 3.216002 1.965321 1.875665 1.235621 1.892003 13.65206 F3 33 3.055541 2.065384 1.802653 1.236654 1.596455 13.56332 F3 34 3.116501 2.105387 1.794685 1.240051 1.792058 13.47669 F3 35 3.120565 1.869322 1.824625 1.189985 1.903255 13.50566 F3 36 3.142652 1.956588 1.778062 1.239856 1.946623 13.51229 F3 37 3.050289 1.899231 1.826452 1.236488 1.885236 13.53656 F3 38 3.10036 2.135655 1.926565 1.424886 1.92556 13.30886 F3 39 3.021056 2.056674 1.881202 1.232301 1.702314 13.59933 F3 40 3.002123 2.092635 1.702188 1.24552 1.95956 13.55699 F3 Table 5. Fisher Criterion for F1–F3 and T-Mode Characteristic Variables. Table 5. Fisher Criterion for F1–F3 and T-Mode Characteristic Variables. Model X C K L F1 and T 0.023192 0.021835 0.003533 0.093898 F2 and T 0.076661 0.017363 3.253002 0.031308 F3 and T 1.369812 0.245968 0.177200 6.105690 Table 6. Diagnostic results of leakage faults in hydraulic cylinder working circuit of different size training samples. Training Samples/Groups C 1 γ 2 Support Vector/Each Test Samples/Groups Misdiagnosed Samples/Groups Accuracy Rate/% 6 32 0.0078125 5 20 2 90 10 1024 0.03125 8 20 2 90 15 512 0.5 14 20 2 90 20 2048 0.5 14 20 1 95 1 penalty coefficient. 2 a parameter of the kernel function. Ni, H.; Lu, L.; Sun, M.; Bai, X.; Yin, Y. Research on Fault Diagnosis of PST Electro-Hydraulic Control System of Heavy Tractor Based on Support Vector Machine. Processes 2022, 10, 791. https://doi.org/10.3390/pr10040791	https://www.mdpi.com/2227-9717/10/4/791
Grow Your Salon Revenue With These Top Tips! - Paperblog Want to boost your revenue this month? Here are some easy ways to get clients in; by using SMS and email campaigns, improving your Online Booking screen, and unleashing the potential of the powerful Client Reconnect tool! Grow Your Salon Revenue With These Top Tips! Want to boost your revenue this month? Here are some easy ways to get clients in; by using SMS and email campaigns, improving your Online Booking screen, and unleashing the potential of the powerfulClient Reconnect tool! Whatever your situation, rest assured that Phorest has the tools to help you createnew revenue streams. Superboost your SMS and Email campaigns There are a wide range of marketing tools, including SMS and email campaigns that you can set up to boost revenue. It’s not just about getting the messaging right, but also about getting the message to theright audience. The average revenue generated from marketing SMS campaigns for Phorest salons in Ireland was €9,082 per salon in Q4 2022. (Minimum 100 texts sent) Use Filters to target the right customers with pinpoint precision Client Filters allow you to create segment groups of your customers using several variables. Check out ourhow-to guideand learn how to get these filters set up. Take this example: Maybe you want to get some of your top spending clients back in, especially those who haven’t visited for a while. In this instance, you could create these new segments and add 2 filters; Top Clients whose spend total is over €175 per visit,andwho have also not visited in the past 150 days. This represents a highly valuable cohort of clientele who you are missing out on revenue from. You can save this as a custom segment and create a targeted campaign for this cohort of customers. This ensures that the right message is getting to the right people. Don’t waste a single penny targeting the wrong people, and get the clients you want back into your salon! Take advantage of ready-made email and SMS templates and add your own personal touch While email is the more cost-effective option,SMS campaigns have a higher open rate. Let’s look at an example of using filters and templates for an effective campaign. Maybe you want to send a special message; ‘Treat Yourself on Your Special Day’, to clients who have their birthdays coming up soon. You could create a custom filter forhigh-spending clientswho havetheir birthday in the month in question.You can edit one of our pre-made templates to add the custom birthday message and send it to those customers only, encouraging them to book online. Improve your Online Booking screen with these simple steps An exceptional customer experience goes a long way, growing both your reputation and revenue simultaneously. Focusing on service information and branding can also contribute to providing a top-class experience. Add detailed service descriptions Whether you are advertising to new or existing customers, you should have detailed descriptions of all your services on your online booking. If customers are unsure of what they are actually choosing, they are much less likely to follow through with the Online Booking process. Don’t lose valuable business at the last hurdle! A good rule of thumb is to approach the description from the customer’s perspective; if you were a customer of your own salon, what would you want to know about this service? Make your Online Booking screen consistent with your branding Brand consistency lets customers recognize your brand across all channels. Your Online Booking is one of the most important ways that you create revenue, so why not ensure it looks like it’s an integral part of your brand? Our online booking checklist lets you see all the vital components to a stand-out online presence, including tips on adding your brand colours to your online booking page. Don’t forget to set up Rebook SMS Rebook SMS is a proven revenue generator for salons. The feature will send SMS to clients prompting them to book online if they failed to make a booking during their last visit. Setup is simple, and the results more than cover the cost of sending such a campaign. You can choose the channel, message, and timeframe you want the SMS Reminders to be sent. On average, 40 new appointments were generated for salons through reminder SMS in Q4 2022. Not only are these 40 slots filled that would have been otherwise empty, but the resulting revenue far outweighs the cost of sending the SMS campaign. Stay in contact with lapsed customers using Client Reconnect Another great way to protect and increase your revenue is to stay in touch with your lapsed customers. Phorest has a bunch of features that help you to do this, such as Client Reconnect, our automated SMS outreach feature. Client Reconnect is the easiest, most effective way to get existing customers back into your salon. Phorest automatically identifies lapsed clients who are overdue a service and gives you the ability to reach out to these clients with the click of a button. You can pre-populate SMS templates for each service with a custom message and link to book online. Client Reconnect directly identifies anyone who clicks to book online from these SMS campaigns, meaning you can see the total value of each Client Reconnect campaign. Additional revenue generated through Client Reconnect was €494 per salon in Q4 2022. Why not try out automatic SMS today and see how much extra revenue you could make? How are you going to use Phorest to grow your salon revenue? Let us know in the comments below or on Instagram.	https://en.paperblog.com/grow-your-salon-revenue-with-these-top-tips-7337458/
Navy Medicine > Navy Marine Corps Public Health Center > About Us > Leadership The official website of the Navy and Marine Corps Public Health Center (NMCPHC) Leadership LEADERSHIP Commander, Navy and Marine Corps Public Health Center CAPT Matthew D. McLean, MC, USN Captain McLean graduated Summa Cum Laude from Elmira College in 1994 with a BA in Biology-Chemistry before matriculating in the combined M.D.-Ph.D. program at SUNY Upstate Medical University in Syracuse, NY. He defended his Ph.D. thesis in the field of cardiac development with honors in November, 1999 and graduated with both degrees in 2001. He received his commission on September 14, 2001 via the Navy Financial Assistance Program and completed his pediatric internship and residency at the Golisano Children’s Hospital at Strong Memorial Hospital, University of Rochester, NY. He reported to Patuxent River Naval Health Clinic in 2004 followed by Naval Hospital Camp Pendleton (NHCP) in 2006. While at NHCP he redesigned the pediatric curriculum for the family practice residency program, and was recognized as teacher of the year before reporting to Naval Medical Center, Portsmouth (NMCP) in 2008 as the Division Head for Pediatric Primary Care and Pediatric GME faculty. He saw pediatrics through the challenging transition to the primary care medical home care model while also serving as the Medical Director for the busiest nursery in the Navy. He served as Vice-President of the Medical Staff from 2013-2014 before his appointment to the Command Executive Board as Director, Healthcare Business from January 2014 through March, 2019. He managed TRICARE benefit delivery for 160,000 beneficiaries and 45,000 operational assigned sailors and developed unique business tools to maximize provider utilization. During his tenure he streamlined referral processes, increased enrollment to the MTF by 15,000 patients and was the primary command liaison to the enhance Multi-Service Market during a challenging time of tri-service market integration. In 2019 he joined the USS KEARSARGE (LHD3) as the Medical Department Head and Senior Medical Officer successfully transitioning it through a difficult maintenance period and basic phase certifications markedly complicated by COVID19 pandemic. Deputy Commander, Navy and Marine Corps Public Health Center CAPT PAUL SCHIERMEIER, MSC, USN After obtaining his Bachelor of Science Degree in Biology from Southern Illinois University in 1995 and a Master of Science Degree in Industrial Hygiene from Central Missouri State University in 1997, he began his career with the Minnesota Department of Health prior to obtaining his commission in 1999. CAPT Schiermeier completed his first assignment at Navy Environmental and Preventive Medicine Unit No. 5. During this tour he received two research awards from the Bureau of Medicine and Surgery forfleet design reconstruction of composite ventilation and engineering-noise controls onboard amphibious ships. Following completion of this tour he reported to Regional Support Organization San Diego in 2001 where he served as the Staff Industrial Hygiene Officer, and received the Chief of Naval Operations award for achievements in afloat occupational safety and health. In 2005 he reported to USS EMORY S. LAND (AS 39) as the command Safety and Environmental Compliance Officer. In 2006 he received the Admiral Vern Clark Safety Award for his accomplishments. Command Master Chief, Navy and Marine Corps Public Health Center HMCM Joseph V. Dennis, USN Master Chief Dennis is a native of Bridgeport, Texas. After high school, he enlisted in the United States Navy in September 1995. He completed Recruit Training at Great Lakes, IL, Hospital Corpsman "A" School at Naval School of Health Sciences San Diego, CA, and Field Medical Service School in Camp Pendleton CA.	https://www.med.navy.mil/Navy-Marine-Corps-Public-Health-Center/About-Us/Leadership/
Sanders v. Nunley, Civil Action No. 1:95cv237-D-D (N.D. Miss. 1/__/1997), Civil Action No. 1:95cv237-D-D. - Federal Cases - Case Law - VLEX 895477331 0: [object Object]. 1: [object Object]. 2: [object Object]. 3: [object Object] Sanders v. Nunley, Civil Action No. 1:95cv237-D-D (N.D. Miss. 1/__/1997), Civil Action No. 1:95cv237-D-D. Court United States District Courts. 5th Circuit. United States District Courts. 5th Circuit. Northern District of Mississippi Parties CARROL ED SANDERS PLAINTIFF v. JOHNNY NUNLEY, LARRY WILSON, BENNY PARRISH, STEVE GRISHAM, DEBORAH GRISHAM and WESTERN SURETY COMPANY DEFENDANTS. Docket Number Civil Action No. 1:95cv237-D-D. Decision Date 01 January 1997 Page 1 CARROL ED SANDERS PLAINTIFF v. JOHNNY NUNLEY, LARRY WILSON, BENNY PARRISH, STEVE GRISHAM, DEBORAH GRISHAM and WESTERN SURETY COMPANY DEFENDANTS. Civil Action No. 1:95cv237-D-D. United States District Court, N.D. Mississippi, Eastern Division. January __, 1997. MEMORANDUM OPINION This action arises from the plaintiff's allegations of wrongdoing and misconduct on the part of Tishomingo County law enforcement officials. By order dated April 18, 1996, this court denied the motion of the defendants Tishomingo County and Benny Parish to dismiss this §1983 action in part upon the ground that the plaintiff's federal law claims are constrained by the Mississippi statute of limitations which governs various intentional torts, Miss.Code Ann. § 15-1-35. Sanders v. Nunley, et al. , Civil Action No. 1:95cv237-D-D (N.D. Miss. Apr. 18, 1996) (Order Denying Defendants' Motions to Dismiss). The defendant Western Surety Company has now moved this court to dismiss it as a party to this action, and relies upon a virtually identical argument. A. As against the plaintiff's federal law claims Because Congress has not provided a statute of limitations Page 2 for civil rights actions brought under § 1983, federal courts adopt the forum state's general personal injury limitations period. The general limitations period is employed instead of a particular limitations period for enumerated intentional torts. Owens v. Okure , 488 U.S. 235 , 249-50, 109 S.Ct. 573 , 581-82, 102 L.Ed.2d 594 (1989); Flores v. Cameron County , 92 F.3d 258 , 271 (5th Cir. 1996); Piotrowski v. City of Houston , 51 F.3d 512 , 514 n. 5 (5th Cir.1995). This court has consistently applied Owens to prevent the application of Miss.Code Ann. § 15-1-35 to claims arising under § 1983, regardless of whether those federal law claims are analogous to state law intentional torts . 1 See , e.g. , Smith v. Luther , Civil Action No. 4:96cv69-D-B (N.D. Miss. Aug. 14, 1996) (Davidson, J.) (Memorandum Opinion and Order Denying Motion to Dismiss); Lightfoot v. Lowndes Co. , Civil Action No. 1:94cv330-D-D (N.D. Miss. Jun 11, 1996) (Davidson, J.) (Memorandum Opinion and Order Denying Motion to Dismiss). Our sister court in the Southern District of Mississippi does so as well. See , e.g. , Gates v. Walker , 865 F.Supp. 1222 , 1229 (S.D. Miss. 1994); Flowers v. Dickens , 741 F.Supp. 112 , 113 (S.D. Miss. 1990). Fifth Circuit decisions are no different. See , e.g. , Gartrell v. Taylor , 981 F.2d 254 , 256 (5th Cir. 1993); Jackson v. Johnson , 950 F.2d 263 , 265 (5th Cir. 1992); James v. Sadler , Page 3 909 F.2d 834 , 836 (5th Cir.1990). In light of the wealth of recent caselaw on the issue stating that the general three-year limitations period is to apply, this court is not convinced by Western Surety's arguments to rule otherwise in this case. 2 To the extent that the plaintiff has alleged federal law civil rights claims against the defendant Western Surety Company, they are not barred by the applicable statute of limitations. 3 To this extent, then, the motion of the defendant Western Surety Company shall be denied. B. As against the plaintiff's state law claims The plaintiff's complaint can be read broadly to assert both state and federal law claims. Under Mississippi law, the plaintiff may sue the defendant Western Surety in a contract action — directly against the bond of Sheriff Nunley for "breach of the sheriff's official duty." See , e.g. , City of Mound Bayou v. Johnson , 562 So. 2d 1212 , 1217 n. 3 (Miss. 1990); U.S.F.&G. v. Mississippi , 182 So. 2d 919 , 923 (Miss. 1966) ("A suit on the surety contract may be maintained although the liability on the contract grows out of a tort.")(emphasis Page 4 added); Alexander v. Casey , 25 So. 2d 709 (Miss. 1946) ("The suit is on the bond .") (emphasis added); Smith v. Smith , 156 Miss. 288, 125 So.825 (1930). The question before this court is the determination of the appropriate statute of limitations on this claim. 1. Miss. Code Ann. § 15-1-35 The primary contention of the defendant Western Surety is that all of the plaintiff's claims are barred by the application of Mississippi's intentional tort statute of limitations, Miss.Code Ann. § 15-1-35. As the undersigned has already determined that the plaintiff's federal law § 1983 claims are not governed by this statute, it only remains for this court to determine if any of the plaintiff's state law claims are so barred.They are not. As a contract action, the plaintiff's claim against Western Surety on the bond is constrained by the statute of limitations for general contract actions: [I]n the absence of a statute to the contrary, the limitation within which an action must be brought on an official bond is that provided by statute for actions on written contracts. Smith , 125 So. at 826; see also Alexander , 25 So. 2d at 709. While the Smith and Alexander decisions are not fresh jurisprudence, the Mississippi Supreme Court has not yet impugned their worth: Smith and Alexander also involve suits against the surety on an official bond. Each holds that the action on such a bond is in effect a suit on a contract and for that reason is Page 5 outside the one-year [intentional tort] statute of limitations. Nothing in today's case affords us an occasion to reconsider or question the limitations period affecting an action against a surety upon an official bond. City of Mound Bayou , 562 So.2d at 1217 n. 3. The defendant has directed this court to no statute of limitations which otherwise applies to actions upon a sheriff's bond, and therefore Mississippi's three year residual statute of limitations applies.Miss. Code Ann. § 15-1-49. The plaintiff timely filed this action within the three year period, and the defendant's motion shall be denied as to this ground. In their rebuttal brief, the defendant Western Surety for the first time argues that if the intentional tort statute of limitations does not apply, then the one year limitations period set forth in Miss.Code Ann. § 15-1-33 does apply. Its failure to raise this matter in its initial motion alone justifies denial of the motion on this ground at this juncture, as it has failed to give the plaintiff an opportunity to respond on this matter. In any event, this...	https://case-law.vlex.com/vid/sanders-v-nunley-civil-895477331
Retail space driving real estate growth in EA as multinationals take up premises - The East African Increasing demand for mall space is luring multinational clients. Retail space driving real estate growth in EA as multinationals take up premises The Hub Karen Mall, Nairobi. Demand for retail space, particularly in Nairobi has been increasing gradually as shopping malls become popular. FILE PHOTO \| NATION Summary Increasing number of multinationals looking for retail space have put East Africa’s property market on the global radar for investment. Demand for retail space, particularly in Nairobi has been increasing gradually as shopping malls become popular. However, the high cost of credit and soaring cost of construction have slowed down investments in the sector, according to a survey by Cytonn Investment Ltd. Increasing demand for houses and an increasing number of multinationals looking for retail space have put East Africa’s property market on the global radar for investment. But even as the region’s real estate sector offers attractive returns to investors, the high cost of credit and soaring cost of construction have slowed down investments in the sector, according to a survey by Cytonn Investment Ltd. The survey, whose report was released last week, shows that although each of the EAC member states has distinct challenges to the growth of the real estate sector, construction costs and cost of credit stand out across Kenya, Uganda, Tanzania and Rwanda. In Rwanda, for instance, construction cost is about 20 per cent higher than Kenya since the country imports most of its construction materials. Inadequate funding has resulted in excessive debt financing, with debt interest rate ranging from 17 per cent to 19 per cent per annum. “Financing for development is not only expensive, but also difficult to access,” says the report. In Kenya the real estate sector slowed down in the first seven months (January-July) of (2017, largely due to the enforcement of the interest rates cap, which prompted banks to reduce lending to the private sector in favour of the government. Increasing demand However the demand for retail space, particularly in Nairobi has been increasing gradually as shopping malls become popular. However, in Kenya real estate has consistently outperformed other investment options such as stocks and bonds in the last five years, generating returns of 25 per cent per annum compared with an average of 12.4 per annum in traditional asset classes. The attraction of international retailers to the market and improving quality of retail stock has enabled landlords to quote substantially higher rents. According to property consulting firm Knight Frank, the retail property segment continues to be a major focus for development activity, causing the shopping mall concept to take root in many sub-Saharan cities. Dilemma facing banks Nairobi has been a retail development hotspot over the past two years, which saw the opening of the Two Rivers Mall, Garden City Mall and The Hub Karen. The real estate sector, which was previously dominated by individual developers, has seen entry of more institutional developers such as Saccos, private equity firms and foreign institutions. The introduction of Real Estate Investment Trusts (Reits) — a financial instrument that provides units of ownership in housing — as a way to raise funding and exit real estate development, is expected to attract more institutional investors. Uganda’s capital markets regulator has beenpushing for more people to take up Reitsto speed up sale of new buildings and widen investment choices for fund managers. “There are about 25 real estate properties available locally, worth $10 million per unit and we need one of them to list before the Reits segment takes off,” Capital Markets Authority chief executive Keith Kalyegira told The EastAfricanearlier in March. A huge housing deficit and low supply of office space created demand for the residential and commercial office units in Kampala, leading to attractive returns of 6.8 per cent and 10.6 per cent, respectively. Uganda has an estimated housing deficit of 1.6 million with Kampala reporting an annual deficit of 100,000 houses. Uganda is currently experiencing a high interest rate environment with banks lending at between 22 per cent and 28 per cent, making it expensive to borrow to construct or buy a house. Austerity measures High costs of construction inputs, high costs of transportation from ports and high land costs in some suburbs, such as Nakasero and Kololo, also raise development costs, reducing the attractiveness and affordability of the housing market. In Tanzania access to credit is a major problem. Access to credit fell from 24.8 per cent in 2015 to 7.2 per cent in 2016 and to 0.3 per cent in 2017 attributable to a rise in non-performing loans. These pushed banks to lend to the government. Some government policies have also hindered investment in property. These include austerity measures like the surplus income cuts for government employees, which restricts their property purchasing capabilities, as well as a strict tax regime since 2015 resulting in reduced spending. It has also resulted in closure of firms and scaling back of multinational firms. Tanzania imposed 18 per cent Value Added Tax on all property purchases, increasing the cost of buying property. In Kenya, VAT is only imposed on commercial real estate purchases. Housing demand is estimated to grow by 200,000 units annually with the cumulative deficit currently at three million units. Its formal retail activity is centred around Dar es Salaam in premium spaces such as Mlimani City Mall, in Mwenge, Aura Mall in Upanga, and Mkuki Mall in Kisutu.	https://www.theeastafrican.co.ke/business/Retail-space-driving-real-estate-growth-in-East-Africa-/2560-4368226-psmneg/index.html
Siyasan Branch Post Office, Idar 50, Sabarkantha, Gujarat Get Siyasan post office address, pincode, phone number, Siyasan speed post tracking, saving scheme and location map. Siyasan Post Office, Idar Siyasan Post Officeis located at Siyasan, Idar, Sabarkanthaof Gujarat state. It is a branch office (B.O.). A Post Office (PO) / Dak Ghar is a facility in charge of sorting, processing, and delivering mail to recipients. POs are usually regulated and funded by the Government of India (GOI). Pin code of Siyasan POis 383450. This Postoffice falls under Sabarkantha postal division of the Gujarat postal circle. The related head P.O. for this branch office is Himatnagar head post office and the related sub-post office (S.O.) for this branch office is Vasai post office. Siyasan dak ghar offers all the postal services like delivery of mails & parcels, money transfer, banking, insurance and retail services. It also provides other services including passport applications, P.O. Box distribution, and other delivery services in Siyasan. The official website fo this PO is http://www.indiapost.gov.in. Types of Post Offices are basically classified into 3 types, namely – Head Post Office, Sub-Post Office including E.D. Sub-Office and Branch Postoffice. Siyasan P.O. is a Branch Post Office. So far as the public is concerned, there is basically no difference in the character of the service rendered by Sub-Post Offices and Head-Post Offices except in regard to a few Post Office Savings Bank (SB) transactions. Certain Sub Post Offices do not undertake all types of postal business. Facilities are generally provided at Branch Post Offices for the main items of postal work like delivery and dispatch of mails, booking of registered articles and parcels accepting SB deposits and effecting SB withdrawals, and issue and payment of money orders, though in a restricted manner. Post Office Type Head Post Office Sub-Post Offices including E.D. Sub-Offices Branch Post Office Siyasan Post Office & Its Pin Code Branch Office Information Siyasan Post Office Services Mail Services Parcels Retail Services Premium Services Speed Post India Post Speed Post Tracking Tracking System India Post Tracking Number Formats Express Parcel Post Media Post Greetings Post Logistics Post ePost Office Financial Services Savings Bank (SB) Account Recurring Deposit (RD) Account Monthly Income Scheme (MIS) Monthly Public Provident Fund (PPF) Time Deposit (TD) Senior Citizen Saving Scheme (SCSS) National Savings Certificate (NSC) Kisan Vikas Patra (KVP) Sukanya Samriddhi Accounts (SSA) Post Office Timings India Post Tracking Siyasan Post Office Recruitment Location Map Contact Details About India Post Siyasan Post Office & Its Pin Code Often Post Offices are named after the town / village / location they serve. The Siyasan Post Office has the Postal Index Number or Pin Code 383450. A Pincode is a 6 digit post code of postal numbering system used by India Post. The first digit indicates one of the regions. The first 2 digits together indicate the sub region or one of the postal circles. The first 3 digits together indicate a sorting / revenue district. The last 3 digits refer to the delivery post office type. P.O. Name Siyasan PO Pincode 383 450 The first digit of 383450 Pin Code '3' represents the region, to which this Post Office of Siyasan belongs to. The first two digits of the Pincode '38' represent the sub region, i.e, Gujarat. The first 3 digits '383' represent the post-office revenue district, i.e, Sabarkantha. The last 3 digits, i.e, '450' represent the Siyasan Delivery Branch Office. Branch Office Information The Siyasan Post Office is a branch office. The Delivery Status for this PO is that it has delivery facility. Postal division name for this Dak Ghar is Sabarkantha, which falls under Ahmedabad Hq region. The circle name for this PO is Gujarat and it falls under Idar Taluka and Sabarkantha District. The state in which this Dakghar is situated or located is Gujarat. The related head postoffice is Himatnagar post office and the related sub post office is Vasai post-office. The phone number of Siyasan post office is unavailable at present. PO Type Branch Office Delivery Status Delivery Postal Division Sabarkantha Postal Region Ahmedabad Hq Postal Circle Gujarat Town / City / Tehsil / Taluka / Mandal Idar District Sabarkantha State Gujarat Related Sub PO Vasai Sub Office Related Head PO Himatnagar Head Post Office Siyasan Post Office Services Traditionally the primary function of Siyasan post office was collection, processing, transmission and delivery of mails but as of today, a Post Office offers many other vital services in addition to its traditional services. The additional services provided by a Dak Ghar include – Mail Services, Financial Services, Retail Services and Premium Services. Mail Services Mail Services are the basic services provided by Siyasan P.O. Mails and mail services include all or any postal articles whose contents are in the form of message which may include Letters, Postcards, Inland letter cards, packets or parcels, Ordinary mails etc. Parcels Mail Service also includes transmission and delivery of Parcels. A parcel can be anything ranging from a single written letter or anything addressed to an addressee. No parcel shall be by any chance be in a shape, way of packing or any other feature, such that it cannot be carried or transmitted by post or cause serious inconvenience or risk. Every parcel (including service parcels) that needs to be transmitted by post must be handed over at the window of the post office. Any parcel found in a letter box will be treated and charged as a registered parcel. Delivery services are provided by some selected delivery and branch post offices. This dakghar have the facility of delivery, thus the people of Siyasan and nearby localities can avail all the types of mail services. Retail Services Post offices in India serve in various ways and Siyasan Post Office offer most of the retail services. They offer the facility to accept or collect constomer bills like telephone or mobile bills, electricity bills for Government and private organizations through Retail Post. Some of the aditional agency services that Post offices offers through retail services are as follows - Telephone revenue collection, e-Ticketing for Road Transport Corporations and Airlines, Sale of UPSC forms, university applications, Sale of Passport application forms, Sale of Gold Coins, Forex Services, Sale of SIM and recharge coupons, Sale of India Telephone cards, e-Ticketing of Railway tickets etc. The postal customers of Siyasan can pay their bills and avail other retail services from this Dak Ghar. Premium Services Most of the premium services can be availed by the Siyasan peoples and nearby living people. The premium services provided by Siyasan Post Office are - Speed Post, Business Post, Express Parcel Post, Media Post, Greeting Post, and Logistics Post. Speed Post Speed Post is a time bound service in express delivery of letters and parcels. The max weight up to which an article or parcel be sent is 35 kgs between any two specified stations in India. Speed Post delivers 'Value for money' to everyone and everywhere, delivering Speed Post upto 50 grams @ INR 35 across the country and local Speed Post upto 50 grams @ INR 15, excluding applicable Service Tax. Kindly check official website for updated Speed Post service charges. India Post Speed Post Tracking Speed Post offers a facility of on-line tracking and tracing that guarantees reliability, speed and customer friendly service. Using a 13 digit barcode that makes a Speed Post consignment unique and identifiable. A web-based technology (www.indiapost.gov.in/speednettracking.aspx) helps the Siyasan customers track Speed Post consignments from booking to delivery. Tracking System Except Speed Post, India Post also allows people to track their order information for certain products like Parcels, Insured letters, Speed Post, Registered Post, Electronic Money Orders (EMO) and Electronic value payable parcel (EVPPs) etc. The tracking number is available on the receipt given at Siyasan Post Office. Using the tracking number postal customers can find out the date and time of dispatch of an article at various locations. The time of booking and the time of delivery of article. India Post Tracking Number Formats Different types of postal service have different kinds of tracking number formats. The tracking number for Express Parcel is a 13 digit alphanumeric format. The format for Express Parcel is XX000000000XX. The tracking number for a Registered Mail is a 13 digit alphanumeric number and its format is RX123456789IN. But a Electronic Money Order (EMO) has a 18 digit tracking number and its format is 000000000000000000. For domestic Speed Post (EMS) there is a 13 digit alphanumeric tracking number with the format EE123456789IN. Bharatiya Dak Ghar Seva Tracking Number Format Number of Digits Electronic Money Order (eMO) 000000000000000000 18 Express Parcel XX000000000XX 13 International EMS Artilces to be delivered in India EE123456789XX 13 Registered Mail RX123456789IN 13 Speed Post (EMS) Domestic EE123456789IN 13 Express Parcel Post In Express Parcel Post, the Siyasan postal customer gets time bound delivery of parcels. These parcels will be transmitted through air or any other fastest mean available at that time. Minimum chargeable weight for which Express Parcel consignments will be booked is 0.5 Kg. Maximum weight of Express Parcel consignments which shall be booked across the Post Office counter by a retail customer shall be 20 Kg and maximum weight that can be booked by corporate customer is 35 kgs. Media Post India Post offers a unique way or concept to help the Indian corporate organisations and the Government organizations reach potential customers through media post. Through media post people can advertise on postcards, letters, aerogramme, postal stationary etc. Customers get to see the logo or message of the respective corporate or government organizations. The Aerogramme even gives the organizations the opportunity to make their product have a global impact. Greetings Post Greeting Post is yet another innovative or unique step by India Post. It consists of a card with an envelope with pre-printed and pre attached postage stamp on the envelope. The stamp on the envelope is a replica of the design that appears upon the card but in miniature form. Thus there is no need affix postage stamps on the envelope implicitly saving your time of going to post offices and standing in the queue. All the rules and that are applicable for the postage dues will also be applicable to the Greeting Post. Logistics Post Logistics Post manages the entire transmission and distribution side of the parcels. It deals with collection of goods, storage of goods, carriage and distribution of the various parcels or goods, from order preparation to order fulfilment. And that too at the minimum possible price. Logistics Post services provides the Siyasan postal customer with cost-effective and efficient distribution across the entire country. ePost Office The advent of internet made communication very rapid through emails. But, the internet has not yet reached most of the rural parts of India. To change this division between rural & urban life, and to get the benefit of internet technology to Siyasan people's lives, Indian Postal Department has introduced e-post. e-post is a service in which personalized handwritten messages of customers are scanned and sent as email through internet. And at the destination address office, these messages are again printed, enveloped and delivered through postmen at the postal addresses. E-post centres are established in the Post Offices, covering a large geographical area including major cities and districts. These e-post centres are well equipped with internet connection, scanners, printers and other necessary hardware equipment. However, this e-post service doesn’t particularly need a e-post centre, but can this facility can be availed at any normal Post Office or you can visit www.epostoffice.gov.in to access postal services on your desktop, laptop or even on mobile. If a message is booked at Siyasan post office, the post is scanned and sent to an e-post centre by e-mail and a mail received at e-post centre is printed and sent to nearby Post Office for dispatch. A Siyasan customer can also avail these services of an e-post, at his/ her home. All he/ she has do is to register as a user at www.epostoffice.gov.in website. After registration, a user can use e-post by scanning and sending messages, printing and receive messages. The message to be scanned must not be written in a paper not more A4. There is no limit for sending number of sheets of messages in e-post. E-Post Office offers certain services like – Philately, Postal Life Insurance, Electronic Indian Postal Order, Information Services, Track & Trace and Complaints & Guidelines services. Philately Philately service deals with collection, sale and study of postage stamps. Philately includes lot of services Philately Information, Stamp issue Program, Stamps List and Buy Stamps service. Postal Life Insurance (PLI) A service offered by the Government to pay a given amount of money on the death of an individual to his prescribed nominee. The amount may also be paid to the person himself, in case he survives that maturity period. The two services offered under Postal Life Insurance are – Pay Premium service and PLI information. Electronic Indian Postal Order eIPO or Electronic Indian Postal Order is a facility to purchase an Indian Postal Order electronically by paying a fee on-line through e-Post Office. This service is launched by the Department of Posts, Ministry of Communications & IT, Government of India. eIPO can now be used by Indian Citizens living in India for paying online fee, whoever seeks information under the RTI Act, 2005. eIPO offers 2 types of services – eIPO information and payment of online fees. Information Services This helps Siyasan customers to get information regarding certain products like – Pin Code search, Speed post, Banking, Insurance, Business Post, Logistics Post, IMTS and many more other services. Track & Trace The track & trace service is very helpful as it aids in getting information of our valuables. Track & Trace service offers 5 different services – Pin Code search, EMO tracking, Speed Post tracking, WNX tracking and International mail service. Complaints & Guidelines Using e-post office service Siyasan postal costumer can access services based on – complaint registration, complaint status and guidelines on complaints. ePost Office Website www.epostoffice.gov.in Financial Services The customers of Siyasan can enjoy the various savings schemes available in this post office that prove to be highly beneficial for the people living in Siyasan area. The Financial service offered by PO includes Savings and Postal Life Insurance (PLI). There are various options available to save and invest with post-offices. The commonly used ones include - Savings account, Recurring Deposit, Monthly Income Scheme, Monthly Public Provident Fund, Time Deposit, Senior Citizen Saving Scheme, National Savings Certificate, Kisan Vikas Patra and Sukanya Samriddhi Yojana. Post Office also offers Insurance product through Postal Life Insurance (PLI) and Rural Postal Life Insurance (RPLI) schemes that offer low premium and high bonus. Post Office Financial Services Kisan Vikas Patra (KVP) Monthly Income Scheme (MIS) Monthly Public Provident Fund (PPF) National Savings Certificate (NSC) Recurring Deposit (RD) Account Savings Bank (SB) Account Senior Citizen Saving Scheme (SCSS) Sukanya Samriddhi Accounts (SSA) Time Deposit (TD) Savings Bank (SB) Account A Savings bank account serves the need of regular deposits for its customers as well as withdrawals. Cheque facility is also avail by Siyasan postal consumers. Recurring Deposit (RD) Account A post office offers a monthly investment option with handsome return at the time period with an option to extend the investment period. Insurance facility is also available with certain conditions. Monthly Income Scheme (MIS) MIS offers a fixed investment technique for five or more years with monthly interest payment to the account holder. There is also a facility of automatic crediting of interest to SB account of the Siyasan postal customer. Monthly Public Provident Fund (PPF) This service offers intermittent deposits subject to a particular limit for a time period of 15 years with income tax exemptions, on the investment. It also offers loan and withdrawal facilities for the postal customers. Time Deposit (TD) Fixed deposit option for periods ranging from one, two, three to five years with facility to draw yearly interest offered at compounded rates. Automatic credit facility of interest to SB account. Senior Citizen Saving Scheme (SCSS) Offers fixed investment option for senior citizens for a period of five years, which can be extended, at a higher rate of interest that are paid in quarterly instalments. National Savings Certificate (NSC) NSC is offered with a fixed investment for 5 or 10 years on certificates of various denominations. Pledging facility available for availing loan from Banks. Kisan Vikas Patra (KVP) Kisan Vikas Patra is a saving certificate scheme in which the amount Invested doubles in 110 months (i.e. 9 years & 2 months). It is available in denominations of Rs 1,000, 5000, 10,000 and Rs 50,000. Minimum deposit is Rs 1000/- and there is no maximum limit. The KVP certificate can be purchased by any adult for himself or on the behalf of a minor. This certificate can also be transferred from one account holder to another and from one post office to another. This certificate can be en-cashed only after 2 and 1/2 years from the date of issue. Sukanya Samriddhi Accounts (SSA) Sukanya Samriddhi Account Yojana offers a small deposit investment for the girl children as an initiative under 'Beti Bachao Beti Padhao' campaign. This yojana is to facilitate girl children proper education and carefree marriage expenses. One of the main benefits of this scheme is that it is very affordable and offers one of the highest interest rates. Currently its interest rate is set as 8.6% per annum that is again compounded yearly. The minimum deposit allowed in a financial year is INR. 1000/- and Maximum is INR. 1,50,000/-. Subsequent deposits can be made in multiples of INR 100/-. Deposits can be made all at a time. No limit is set on number of deposits either for a month or a financial year. A legal Guardian can open an account in the name of a Girl Child. Account can be closed only after completion of 21 years of the respective child. The normal Premature closure allowed is after completion of 18 years only if that girl is getting married. Post Office Timings The official working hours of Post Offices vary from one another, but the general Post Office opening time starts from 08:00 AM or 09:00 AM or 10:00 AM and the closing time is 04:00 PM or 05:00 PM or 06:00 PM respectively. The working days are from Monday to Saturday, Sunday being a holiday. This doesn't include the public holidays or the extended working hours. You can verify the working hours of Siyasan Branch Post Office from the official resources. India Post Tracking Online tracking of India Post allowed Siyasan people to access their postal article tracking information and confirm the delivery of their postal article by using the tracking number assigned to them at the time of Booking. They can find the tracking number on the Postal acknowledgement handed over to them at the Siyasan Branch Post Office counter at the time of postal article booking. Following items can track through the www.indiapost.gov.in/articleTracking.aspx official website. Business Parcel Business Parcel COD Electronic Money Order (e-MO) Electronic Value Payable Parcel (eVPP) Express Parcel Express Parcel COD Insured Letter Insured Parcel Insured Value Payable Letter Insured Value Payable Parcel International EMS Registered Letter Registered Packets Registered Parcel Registered Periodicals Speed Post Value Payable Letter Value Payable Parcel The India Post tracking system is updated at regular intervals to give the Siyasan postal customers with the most up to date information available about the location and status of their postal article. They'll be able to find out the following: When their postal article was booked When their postal article was dispatched at various locations during its Journey When their postal article was received at various locations during its Journey When their postal article was delivered, or When a delivery intimation notice was issued to notify the recipient that the postal article is available for delivery Siyasan Post Office Recruitment For latest Siyasan post office recruitment kindly visit www.indiapost.gov.in/recruitment.aspx. Location Map Siyasan Branch Post Office is located in Siyasan, Idar, Sabarkantha. Contact Details All the queries or complaints regarding Bill Mail Service, Booking Packets, Business Post, Direct Post, Flat Rate Box, Indian Postal Orders, Inland Letters, Instant Money Orders, Insurance of Postal Articles, Insurance of Postal Parcels, Letters, Logistics Posts, MO Videsh, Money Orders, Parcels, Post Office Savings Bank, Postal Life Insurance, Postcards, Registration of Postal Articles, Registration of Postal Parcels, Rural Postal Life Insurance, Saving Certificates, Small Saving Schemes, Speed Post, Value Payable Post etc.services in Siyasan Post Office, can be resolved at Siyasan Branch Post Office. You can send letters to "Postmaster, Siyasan Branch Post Office, Siyasan, Idar, Sabarkantha, Gujarat, India, Pincode: 383 450". The official website of the Berhampur University Sub Office is http://www.indiapost.gov.in. Siyasan Branch Office Address: Siyasan Branch Post Office, Siyasan, Idar, Sabarkantha , Gujarat , India Pin Code: 383450 Website: www.indiapost.gov.in About India Post India Postis a government-operated postal system, which is part of the Ministry of Communications and Information Technology of the Government of India. It has the largest Postal Network in the Indiawith over 154882 Post Offices. There are around 139182 Post Offices in the rural India and 15700 Post Offices in urban India. The individual post office serves an area of 21.22 Sq. Km. and a population of 8221 people. The slogan of India Post is Dak Seva Jan Seva. There are 25464 departmental post offices and 129418 extra-departmental branch post offices in India. Siyasan Post Office Summary Dak Ghar Name Siyasan Branch Post Office Pincode 383450 Dakghar Type Branch Office Post Office Delivery Status Delivery Branch Office Postal Division Sabarkantha Postal Region Ahmedabad Hq Postal Circle Gujarat Location Siyasan Town / City / Tehsil / Taluka / Mandal Idar District Sabarkantha State Gujarat Country India Related Sub Office Vasai Sub Office Related Head Office Himatnagar Head Post Office Website www.indiapost.gov.in ePost-office Web Site Address www.epostoffice.gov.in Speed Post Tracking Website www.indiapost.gov.in/speednettracking.aspx Recruitment Web Site Address www.indiapost.gov.in/recruitment.aspx Previous Dak Ghar Kishangadh Post Office Other Post Offices	https://www.postoffices.co.in/gujarat-gj/siyasan-idar-50-sabarkantha/
Pembrolizumab in Treating Patients With Intermediate or High-Risk Smoldering Multiple Myeloma - Full Text View - ClinicalTrials.gov Pembrolizumab in Treating Patients With Intermediate or High-Risk Smoldering Multiple Myeloma ClinicalTrials.gov Identifier: NCT02603887 Recruitment Status : Active, not recruiting First Posted : November 13, 2015 Results First Posted : March 20, 2020 Last Update Posted : March 29, 2023 Sponsor: National Cancer Institute (NCI) Information provided by (Responsible Party): M.D. Anderson Cancer Center Study Details Study Description Brief Summary: This pilot early phase I trial studies pembrolizumab in treating patients with slow growing (smoldering) multiple myeloma with intermediate or high-risk of spreading. Immunotherapy with monoclonal antibodies, such as pembrolizumab, may help the body's immune system attack the cancer, and may interfere with the ability of tumor cells to grow and spread. Condition or disease Intervention/treatment Phase Smoldering Plasma Cell Myeloma Other: Laboratory Biomarker Analysis Biological: Pembrolizumab Early Phase 1 Detailed Description: PRIMARY OBJECTIVE: I. To determine the overall response rate after 8 cycles of treatment according to the International Myeloma Working Group Criteria (IMWG). SECONDARY OBJECTIVES: I. To determine time to progression to multiple myeloma (TTP) at 30 months from study entry. II. To determine overall survival (OS). III. To determine duration of response (DOR). IV. To determine the clinical benefit rate (CBR) after 8 cycles of treatment according to the modified IMWG Criteria for multiple myeloma (MM). V. To evaluate safety and tolerability of single agent treatment in this population. I. Rate of minimal residual disease (MRD) negativity at complete remission (CR). II. Molecular profiling (including whole exome sequencing and gene expression profiling) and cellular (including flow cytometry) profiling at baseline and/or at progression using bone marrow aspirate samples and peripheral blood. III. Immunophenotypic characterization of dendritic, T-, B-, natural killer (NK)- and natural killer T (NKT)-cells, and inhibitory/activation markers on tumor cells at baseline and at completion of 8 cycles of therapy in bone marrow aspirate samples and/or peripheral blood. IV. Evaluation of changes in PD-L1 and PD-1 expression at baseline/end of 8 cycles of treatment and correlate with clinical response. OUTLINE: Patients receive pembrolizumab intravenously (IV) over 30 minutes on day 1. Treatment repeats every 21 days for up to 24 courses in the absence of disease progression or unacceptable toxicity. After completion of study treatment, patients are followed up every 6-12 months. Study Design Layout table for study information Study Type : Interventional (Clinical Trial) Actual Enrollment : 20 participants Allocation: N/A Intervention Model: Single Group Assignment Masking: None (Open Label) Primary Purpose: Treatment Official Title: Pilot Single Arm, Single Center, Open Label Trial of Pembrolizumab in Patients With Intermediate and High Risk Smoldering Multiple Myeloma Actual Study Start Date : July 20, 2016 Actual Primary Completion Date : December 30, 2017 Estimated Study Completion Date : December 31, 2023 Resource links provided by the National Library of Medicine MedlinePlus Genetics related topics: Multiple myeloma MedlinePlus related topics: Multiple Myeloma Drug Information available for: Pembrolizumab Genetic and Rare Diseases Information Center resources: Multiple Myeloma U.S. FDA Resources Arms and Interventions Arm Intervention/treatment Experimental: Treatment (pembrolizumab) Patients receive pembrolizumab IV over 30 minutes on day 1. Treatment repeats every 21 days for up to 24 courses in the absence of disease progression or unacceptable toxicity. Other: Laboratory Biomarker Analysis Correlative studies Biological: Pembrolizumab Given IV Other Names: Keytruda Lambrolizumab MK-3475 SCH 900475 Outcome Measures Primary Outcome Measures : Overall Response Rate (ORR) [ Time Frame: From cycle 1 to cycle 8, up to 168 days ] Overall response rate is defined as patients who have achieved partial response or higher as described per IMWG criteria for multiple myeloma after 8 cycles of therapy with pembrolizumab. Will be measured according to International Myeloma Working Group Criteria (IMWG) criteria. Response rate will be estimated accordingly. Each cycle has a duration of 21 days. Secondary Outcome Measures : Number of Participants That Had Progression to Multiple Myeloma [ Time Frame: At 30 months from study entry ] Progressive disease assessed by IMWG criteria for multiple myeloma after 8 cycles of therapy with pembrolizumab. Clinical Benefit Rate [ Time Frame: From cycle 1 to cycle 8, up to 168 days ] Minor response or better assessed by IMWG criteria for multiple myeloma after 8 cycles of therapy with pembrolizumab Overall Survival [ Time Frame: Time of start of treatment to death from any cause up to 2 years and 6 months ] Participants in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease Eligibility Criteria Information from the National Library of Medicine Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies. Layout table for eligibility information Ages Eligible for Study: 18 Years and older (Adult, Older Adult) Sexes Eligible for Study: All Accepts Healthy Volunteers: No Criteria Inclusion Criteria: Adult patients with intermediate or high-risk smoldering multiple myeloma (SMM) are eligible; patients need to have clonal bone marrow plasma cells >= 10% and/or monoclonal spike in blood of >= 3 g/dL and/or monoclonal urine component (Bence jones proteinuria) >= 500 mg/24 hours and need to meet subject inclusion criteria and exclusion criteria as per below Patients must have histologically confirmed SMM based on the following criteria: (A) Mayo clinic criteria (patient must have at least 2 risk factors present): 1. Bone marrow core biopsy plasma cell involvement by cluster of differentiation (CD)138 immunohistochemistry >= 10% 2. Monoclonal spike >= 3 g/dL 3. Free light chain ratio in serum < 0.125 or > 8; 2 of 3 risk factors: intermediate risk for progression at a rate of 51% at 5 years, 3 of 3 risk factors: high risk for progression at a rate of 76% at 5 years OR (B) Programa para el Tratamiento de Hemopatias Malignas (PETHEMA) criteria (patient must have at least 1 risk factor present) 1. >= 95% abnormal plasma cells/total plasma cells in bone marrow compartment 2. Immunoparesis 1 of 2 risk factors: intermediate risk for progression at a rate of 46% at 5 years, 2 of 2 risk factors: high risk for progression at a rate of 72% at 5 years OR (C) Southwestern Oncology Group (SWOG) criteria (patient must have 2 risk factors present or one risk factor if this risk factor is a 70-gene signature (GEP70) score of > 37.2) 1. Monoclonal spike >= 3 g/dL 2. Involved free light chain >= 25 mg/dL 3. GEP70 risk score > 37.2 >= 2 risk factors: high risk of progression at a rate of 70% at 2 years; we would also include patients with 1 risk factor as long as this risk factor is GEP70 risk score > 37.2 since patients with this risk factor have an intermediate risk of progression at a rate of 50% at 2 years Creatinine clearance >= 50 ml/min; creatinine clearance (CrCl) will be calculated by Cockcroft-Gault method Eastern Cooperative Oncology Group (ECOG) performance status 0-1 Absolute neutrophil count (ANC) >= 1.0 x 10^9 /L Hemoglobin >= 10 g/dL Platelet count >= 50 x 10^9/L Bilirubin < 1.5 x the upper limit of normal (ULN) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 3.0 x ULN Subjects must be able to give informed consent Female subject of childbearing potential should have a negative urine or serum pregnancy within 72 hours prior to receiving the first dose of study medication; if the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required Female subjects of childbearing potential should be willing to use 2 methods of birth control or be surgically sterile, or abstain from heterosexual activity for the course of the study through 120 days after the last dose of study medication; subjects of childbearing potential are those who have not been surgically sterilized or have not been free from menses for > 1 year Male subjects should agree to use an adequate method of contraception starting with the first dose of study therapy through 120 days after the last dose of study therapy Exclusion Criteria: Evidence of myeloma defining events or biomarkers of malignancy due to underlying plasma cell proliferative disorder meeting at least one of the following 1) Hypercalcemia: serum calcium > 0.25 mmol/L (> 1 mg/dL) higher than the upper limit of normal or > 2.75 mmol/L (> 11 mg/dL) 2) Renal insufficiency: creatinine clearance < 40 ml/min or serum creatinine > 2 mg/dL 3) Anemia: hemoglobin value < 10 g/dL or 2 g/dL < normal reference 4) Bone lesions: one or more osteolytic lesions on skeletal radiography, computerized tomography (CT) or 2-deoxy-2[F-18] fluoro-D-glucose positron emission tomography CT (PET-CT) 5) Clonal bone marrow plasma cell percentage >= 60% 6) Involved: uninvolved serum free light chain ratio >= 100 measured by Freelite assay (The Binding Site Group, Birmingham, United Kingdom [UK]) 7) > 1 focal lesions on magnetic resonance imaging (MRI) studies (each focal lesion must be 5 mm or more in size), if clinically indicated Prior or concurrent systemic treatment for SMM; a) bisphosphonates are permitted; b) treatment with corticosteroids is not permitted; c) radiotherapy is not permitted; d) prior treatment for smoldering multiple myeloma with chemotherapy agents approved for the treatment of multiple myeloma is not permitted Plasma cell leukemia Pregnant or lactating females; breastfeeding should be discontinued if the mother is treated with pembrolizumab Has a diagnosis of immunodeficiency or is receiving systemic steroid therapy or any other form of immunosuppressive therapy within 7 days prior to the first dose of trial treatment Has a known history of active TB (bacillus tuberculosis) Hypersensitivity to pembrolizumab or any of its excipients Has had a prior anti-cancer monoclonal antibody (mAb) within 4 weeks prior to study day 1 or who has not recovered (i.e., =< grade 1 or at baseline) from adverse events due to agents administered more than 4 weeks earlier Has a known additional malignancy that is progressing or requires active treatment; exceptions include basal cell carcinoma of the skin or squamous cell carcinoma of the skin that has undergone potentially curative therapy or in situ cervical cancer Has active autoimmune disease that has required systemic treatment in the past 2 years (i.e. with use of disease modifying agents, corticosteroids or immunosuppressive drugs); replacement therapy (eg., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment Has a history of (non-infectious) pneumonitis that required steroids or current pneumonitis Has an active infection requiring systemic therapy Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial Is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the trial, starting with the pre-screening or screening visit through 120 days after the last dose of trial treatment Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-programmed death ligand 2 (PD-L2) agent Has a known history of human immunodeficiency virus (HIV) (HIV 1/2 antibodies) Has known active hepatitis B (e.g., hepatitis B surface antigen [HBsAg] reactive) or hepatitis C (e.g., hepatitis C virus [HCV] ribonucleic acid [RNA] [qualitative] is detected) Has received a live vaccine within 30 days of planned start of study therapy; Note: seasonal influenza vaccines for injection are generally inactivated flu vaccines and are allowed; however intranasal influenza vaccines (e.g., Flu-Mist) are live attenuated vaccines, and are not allowed Evidence of interstitial lung disease National Cancer Institute (NCI) Investigators Layout table for investigator information Principal Investigator: Elisabet E Manasanch, MD M.D. Anderson Cancer Center Study Documents (Full-Text) Documents provided by M.D. Anderson Cancer Center: Study Protocol and Statistical Analysis Plan [PDF] April 8, 2016 Additional Information: MD Anderson Cancer Center Publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number): Manasanch EE, Han G, Mathur R, Qing Y, Zhang Z, Lee H, Weber DM, Amini B, Berkova Z, Eterovic K, Zhang S, Zhang J, Song X, Mao X, Morgan M, Feng L, Baladandayuthapani V, Futreal A, Wang L, Neelapu SS, Orlowski RZ. A pilot study of pembrolizumab in smoldering myeloma: report of the clinical, immune, and genomic analysis. Blood Adv. 2019 Aug 13;3(15):2400-2408. doi: 10.1182/bloodadvances.2019000300. Erratum In: Blood Adv. 2019 Sep 24;3(18):2712. Layout table for additonal information Responsible Party: M.D. Anderson Cancer Center ClinicalTrials.gov Identifier: NCT02603887 History of Changes Other Study ID Numbers: 2015-0371 NCI-2015-02135 ( Registry Identifier: CTRP (Clinical Trial Reporting Program) ) 2015-0371 ( Other Identifier: M D Anderson Cancer Center ) First Posted: November 13, 2015 Key Record Dates Results First Posted: March 20, 2020 Last Update Posted: March 29, 2023 Last Verified: March 2023 Layout table for additional information Studies a U.S. FDA-regulated Drug Product: Yes Studies a U.S. FDA-regulated Device Product: No	https://clinicaltrials.gov/ct2/show/NCT02603887
Inorganics \| Free Full-Text \| Gold(III) Pyridine-Benzimidazole Complexes as Aquaglyceroporin Inhibitors and Antiproliferative Agents Gold compounds have been proven to be novel and versatile tools for biological applications, including as anticancer agents. Recently, we explored the potential of Au(III) complexes with bi-dentate N-donor ligands as inhibitors of the membrane water and glycerol channels aquaporins (AQPs), involved in different physiological and pathophysiological pathways. Here, eight new Au(III) complexes featuring a pyridine-benzimidazole scaffold have been synthesized and characterized via different methods. The stability of all the compounds in aqueous solution and their reactivity with glutathione have been investigated by UV–visible spectroscopy. The Au(III) compounds, tested for their AQPs inhibition properties in human Red Blood Cells (hRBC), are potent and selective inhibitors of AQP3. Furthermore, the compounds’ antiproliferative effects have been studied in a small panel of human cancer cells expressing AQP3. The complexes show only very moderate anticancer effects in vitro and are mostly active against the melanoma A375 cells, with marked expression of AQP3 at the level of the nuclear membrane. In general, the AQP3 inhibition properties of these complexes hold promises to develop them as chemical probes to study the function of this protein isoform in biological systems. Gold(III) Pyridine-Benzimidazole Complexes as Aquaglyceroporin Inhibitors and Antiproliferative Agents Brech Aikman 1,† , Margot N. Wenzel 1,† , Andreia F. Mósca 2,† , Andreia De Almeida 1,3 , Wim T. Klooster 4 , Simon J. Coles 4 , Graça Soveral 2,* and Angela Casini 1,* Inorganics 2018 , 6 (4), 123; https://doi.org/10.3390/inorganics6040123 Received: 11 October 2018 / Revised: 14 November 2018 / Accepted: 15 November 2018 / Published: 20 November 2018 (This article belongs to the Special Issue Unconventional Anticancer Metallodrugs and Strategies to Improve their Pharmacological Profile ) Abstract : Gold compounds have been proven to be novel and versatile tools for biological applications, including as anticancer agents. Recently, we explored the potential of Au(III) complexes with bi-dentate N-donor ligands as inhibitors of the membrane water and glycerol channels aquaporins (AQPs), involved in different physiological and pathophysiological pathways. Here, eight new Au(III) complexes featuring a pyridine-benzimidazole scaffold have been synthesized and characterized via different methods. The stability of all the compounds in aqueous solution and their reactivity with glutathione have been investigated by UV–visible spectroscopy. The Au(III) compounds, tested for their AQPs inhibition properties in human Red Blood Cells (hRBC), are potent and selective inhibitors of AQP3. Furthermore, the compounds’ antiproliferative effects have been studied in a small panel of human cancer cells expressing AQP3. The complexes show only very moderate anticancer effects in vitro and are mostly active against the melanoma A375 cells, with marked expression of AQP3 at the level of the nuclear membrane. In general, the AQP3 inhibition properties of these complexes hold promises to develop them as chemical probes to study the function of this protein isoform in biological systems. Keywords: Gold(III) complexes ; pyridine benzimidazole ; aquaporins ; cancer ; stopped-flow spectroscopy ; antiproliferative activity Graphical Abstract 1. Introduction The severe side effects associated with chemotherapy necessitate the development of improved anticancer therapies. Specifically, the discovery of compounds that can disrupt cancerous cellular machinery by novel mechanisms of action is nowadays the focus of intense research. For example, metal-based compounds acting via the interaction with proteins and secondary DNA structures, as well as by alteration of the intracellular redox balance, have become prominent experimental therapeutic agents. Among them, gold complexes have attracted attention in the last years and numerous families of Au(I) and Au(III) compounds have been synthesized and studied for their anticancer properties in vitro and in vivo [ 1 , 2 ]. Overall, the investigation of the cytotoxic activity and related mode of action of cytotoxic gold-based complexes has enabled the identification of their preferential “protein targets”, as it is increasingly evident that DNA is not the unique or major target for such compounds [ 3 ]. In this context, coordination cytotoxic Au(III) compounds have been identified as selective inhibitors of the membrane water channels aquaporins (AQPs) [ 1 , 4 ]. Among the 13 mammalian AQPs described so far, three sub-groups can be recognized based on permeability features: orthodox aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8), which are primarily water selective and facilitate water movement across cell membranes in response to osmotic gradients [ 5 ]; aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10), facilitating the permeation of small uncharged solutes such as glycerol [ 6 ]; and unorthodox aquaporins (AQP11, AQP12), found in intracellular membranes and with reported permeability to water and glycerol [ 7 , 8 , 9 ]. Specifically, the aquaglyceroporins regulate the glycerol content in the epidermis, fat and other tissues and appear to be involved in skin hydration, cell proliferation, fat metabolism, and carcinogenesis. Several studies showed that AQPs are closely associated with cancer proliferation and invasion, and are expressed in at least 20 human cancers [ 10 ]. Moreover, AQPs expression is related to tumour types, grades, proliferation, migration and angiogenesis, rendering these transport proteins attractive as both diagnostic and therapeutic targets in cancer [ 10 ]. To validate the various roles of AQPs in health and disease, and to develop AQP-targeted therapies, the use of selective inhibitors in addition to genetic approaches, holds great promise. However, so far, no reported organic small-molecule AQPs inhibitor possesses sufficient isoform selectivity to be a good candidate for clinical development [ 11 ]. A few years ago, we reported on the potent and selective inhibition of human AQP3 by a series of Au(III) complexes with bidentate N^N ligands [ 12 , 13 ], which could potently and selectively inhibit glycerol permeation through hAQP3 in human red blood cells (hRBC). The most effective inhibitor of the series, Auphen ([Au(phen)Cl 2 ]Cl, phen = 1,10-phenanthroline) had an IC 50 of 0.8 ± 0.08 μM [ 12 ]. In a further study, Auphen’s capacity of inhibiting cell proliferation was examined in various cell lines, including cancerous ones, with different levels of AQP3 expression, and showed a direct correlation between AQP3 expression levels and the inhibition of cell growth by the Au(III) compound [ 14 ]. AQP3 inhibition was also demonstrated in the cell lines where proliferation was mostly affected by treatment with the gold complex [ 14 ]. Structure–activity relationships to optimize the design of AQP3 inhibitors were then established investigating other Au(III) compounds with different N^N ligand scaffolds [ 13 ]. Pursuing the design of more potent and selective AQP3 inhibitors, we have recently observed that the cationic complex [Au(pbzMe)Cl 2 ]PF 6 ( C1 , pbzMe = 1-methyl-2-(pyridin-2-yl)-benzimidazole) is even more efficient than Auphen in inhibiting glycerol permeation via AQP3 [ 1 ], and ca. three orders of magnitude more effective than the neutral related complex [Au(pbzH)Cl 2 ] ( C10 , pbzH = 2-(pyridin-2-yl)-benzimidazole) [ 15 ]. Combined molecular dynamics (MD) and density functional theory (DFT) studies were able to show that C1 , upon binding to Cys40 in AQP3, is able to induce protein conformational changes, leading to the shrinkage of the channel, and thus, preventing glycerol and water permeation [ 15 ]. Following these promising results, we have synthesized a new series of Au(III) complexes based on the 2-(2-pyridyl)benzimidazole (pbzH) N-donor ligand, which is also known to inhibit hepatic enzymes, [ 16 ] and exhibits anticancer activities per se [ 17 ]. In general, metal complexes based on 2-(2′-pyridyl)benzimidazole scaffolds have attracted attention in various established and potential application areas, including medicinal inorganic chemistry [ 18 , 19 , 20 ]. Thus, we report here on the synthesis and characterization of eight new cationic Au(III) derivatives with functionalization at the non-coordinated benzimidazole nitrogen. In addition, two neutral complexes featuring extended aromatic scaffolds (namely pyrene and anthracene), endowed with luminescence properties, have been obtained. The compounds have been tested for their AQPs inhibition properties in human Red Blood Cells (hRBC) using a stopped-flow method, and their effects compared to C1 [Au(pbzMe)Cl 2 ]PF 6 and C10 [Au(pbzH)Cl 2 ]. Furthermore, the compounds’ antiproliferative effects have been studied in a small panel of human cancer cells with different levels of AQP3 expression. 2. Results 2.1. Synthesis and Characterization of Au(III) Complexes The library of functionalised pyridylbenzimidazole ligands L1 – L9 has been obtained by nucleophilic substitution on the non-coordinated nitrogen atom of the commercially available pyridylbenzimidazole by reaction with a halogenated substituent (R–X) in the presence of a base ( Scheme S1, Supplementary Materials ) [ 21 ]. Several types of functional groups have been envisaged to study the influence of both the steric hindrance and the electronic effect on the biological properties of the final gold complexes. In parallel, two additional ligands ( L11 – L12 ) featuring luminescent properties [ 22 ] have also been synthesized with the idea to monitor their fate in cancer cells by fluorescence microscopy ( Scheme S1, supplementary materials ). The use of ligands L1 – L9 , which possess a functionalised amine, gives rise to Au(III) cationic complexes (corresponding C1 – C9 ) by reaction between an equimolar amount of L1 – L9 and NaAuCl 4 , in the presence of an excess of KPF 6 ( Scheme 1 , top). The pure cationic gold complexes can then easily be isolated following precipitation, washing and filtration. On the other hand, reaction between ligands L10 – L12 and NaAuCl 4 in the presence of a base leads to the formation of the neutral complexes C10 – C12 ( Scheme 1 , bottom) [ 15 ]. The identity and the purity of the complexes C1 – C12 was confirmed by NMR, IR and UV–Visible spectroscopies, as well as by mass spectrometry and in some cases by elemental analysis (See Experimental and Supplementary Materials for details, Figures S1–S24 ). The obtained results confirmed the purity of the compounds, which were all obtained in good yields. While all ligands were found to be soluble in most organic solvents, and thus, their NMR analysis was performed in CDCl 3 , the complexes were insoluble in most cases, except in rare examples in acetone or acetonitrile. To ensure a similar analysis for all complexes, their NMR spectra were recorded in DMSO- d 6 . The 1 H NMR spectra of the ligands were easily attributable and the most downfield shifted signals corresponded to the benzimidazole ring. In most cases, the signals of the protons of the pyridyl were found overlapping each other, in addition to the signals of the phenyl rings for the R substituents in the case of L4 – L9 . The 1 H NMR spectra of the complexes were similar to those of the corresponding ligands in terms of number of resonance signals; however, some signals (H a , H c and H h , see Scheme 2 for the numbering scheme) were clearly more affected by the presence of the Au(III)Cl 2 fragment [Δδ (δ coord − δ free ) of 0.24 and 0.47 ppm]. The NMR analysis of both the ligands L11 – L12 and their corresponding complexes C11 and C12 was very challenging due to the electronic similarity and thus proximity on the spectra. However, the number and nature of the signals were compatible with the structures, and further analytical methods allowed us to confirm the purity of the compounds (ESI-MS, UV–Visible spectroscopy and IR). IR analysis of the complexes showed in all cases the presence of C=C bending, C–H stretching and C–N stretching bands, and confirmed the presence of specific chemical groups on the main scaffold: alkyl chains in the case of C1 – C3 , ester group in the case of C5 and C–F bonds for C6 – C9 . Crystals suitable for X-ray diffraction were obtained for complex C6 by slow diffusion of pentane in a concentrated solution of the complex in a mixture of acetonitrile and dichloromethane at room temperature (see Supplementary Materials for details). The structure confirmed a bidentate coordination mode of the ligand L6 onto the gold centre via the nitrogen of the pyridine and the benzimidazole rings, giving rise to square planar complexes. The 4-trifluoromethylbenzyl functional group added on the benzimidazole moiety always points out of the plane, as already described with similar ligands and copper complexes [ 18 ]. It is worth mentioning that the slow process of crystallisation (10–15 days) may favour the partial decomposition of the complexes, specifically the de-coordination of the gold centre and the exchange of counter anions. In fact, the structure of C6 revealed the presence of AuCl 4 counterions in the lattice (see Supplementary Materials ). Furthermore, we attempted to crystallize compound C7 in the same conditions, but the resulting X-ray structure confirmed the de-coordination of the gold centre from one of the nitrogens of the pyridine ligand, thus leading to a neutral gold complex with three coordinated chlorido ligands (see supplementary materials ). The complexes C1 – C12 and their corresponding ligands L1 – L12 have also been investigated for their photophysical properties (see Figures S25–S48 ). Both ligands L1 – L10 and complexes C1 – C9 exhibit a strong absorption band centred around 310–315 nm which can be attributed to π→π* transitions and/or ligand-to-metal charge transfers (LMCT) in the case of the Au(III) complexes. The absorption spectra of ligands L11 – L12 and corresponding complexes C11 – C12 , with the extended conjugated systems, show several bands attributed to the same transitions between 330 and 390 nm. Ligands L1 – L10 and complexes C1 – C9 all have single fluorescence emission bands centred around 375–380 nm, representing a Stokes shift of about 65 nm. Complexes C11 – C12 and their corresponding ligands possess extended aromatic and conjugated systems; thus, a shift in the emission bands is observed: ligand L11 and complex C11 emit at 415 nm whereas L12 and C12 exhibit an emission band around 450 nm ( Figures S45–S48 ). The quantum yield of fluorescence (Φ F ) has also been assessed for all the reported compounds (see Figures S25–S48 ). While the ligands with the alkane substituents ( L1 – L3 ) have relatively high quantum yields (50–60%), the ligands with the functionalised benzyl groups ( L4 – L9 ) have decreased quantum yields between 27 and 42%. The ligands with the extended aromatic systems L11 and L12 have quantum yields of 74 and 61%, respectively ( Figures S45 and S47 ). In general, upon coordination of the ligands to the gold(III) ion, almost all quantum yields of luminescence are decreased due to the “heavy metal effect”, with the exception of complex C12 (Φ F = 71%). 2.2. UV–Visible Stability Studies The stability of the gold complexes was investigated using UV–Visible spectroscopy before further biological testing. Thus, the absorbance of the compounds’ solutions in PBS buffer (pH 7.4) was measured between 300 and 800 nm at regular time intervals during 24 h at room temperature, allowing the monitoring of possible compound’s transformations such as hydrolysis, reduction and/or precipitation. In parallel, as Au(III) complexes tend to be reduced in physiological conditions to Au(I) and even Au(0), the reactivity of the compounds with the intracellular reducing agent glutathione (GSH) was monitored in the same conditions. All the Au(III) compounds exhibit intense transitions in the 300–400 nm range, characteristic of the Au(III) chromophore, that may be straightforwardly assigned as LMCT bands. Complexes C1 , C2 , C5 , C6 and C9 were found to be mostly stable over the first 6 h in PBS buffer (pH 7.4) with no significant change in the UV–Visible spectra ( Figures S49, S50, S53, S54 and S57 ). The observed small spectral changes developing with time might be related to the occurrence of partial hydrolysis processes. Instead, the spectra of complexes C3 , C4 , C7 , C8 and C10 were found to undergo major changes over the first few hours ( Figures S51–S52, S55–S56 and S58 ), leading to the disappearance of the classical LMCT bands, suggesting disruption of the Au(III) complex. Complexes C11 – C12 were moderately stable in solution ( Figures S59 and S60 ) featuring hypocromic shifts in their spectra over the first 6 h of incubation. In the presence of GSH, all the compounds immediately reacted to form adducts leading to the loss of the N-donor ligands from the gold centre, as demonstrated by the disappearance of the LMCT bands ( Figures S49–S58 ). Only compounds C11 and C12 were scarcely reactive and maintained their spectral features over time after the addition of GSH ( Figures S59 and S60 ). 2.3. Inhibition of Aquaporins Based on the previosuly discussed stability studies, only the most stable gold complexes were tested for their AQP1 and AQP3 inhibition properties in hRBC by stopped-flow spectroscopy, according to previously reported procedures [ 12 ]. A representative IC 50 curve for the inhibition of glycerol permeation via AQP3 by two gold compounds is reported in Figure 1 . The obtained results are summarized in Table 1 and show that all the new cationic complexes C2 , C4 , C5 , C6 , C9 and C11 are able to selectively inhibit glycerol permeation via AQP3, with IC 50 values in the sub-micromolar level, comparable to C1 and Auphen. The antiproliferative activities of the corresponding ligands are presented in the supplementary ( Table S1 ). The most effective compound was C6 . In line with previous results, the neutral compound C10 was ca. one order of magnitude less effective as an AQP3 inhibtior [ 15 ]. Of note, all the compounds were inactive as inhibitors of the orthodox water channel AQP1 in the same cellular model (data not shown). 2.4. Expression of AQP3 in Cancer Cells and Antiproliferative Activities Afterward, the compounds—with the exception of the highly unstable C7 and C8 —were evaluated for their antiproliferative activities against a panel of human cancer cells in vitro. The cells were selected and studied for their level of expression of human AQP3. Information on the expression of AQP3 in cancer lines is limited and most of the data currently available refers to mRNA expression levels [ 23 ]. Specifically, cells were studied for the expression levels of human AQP3 by flow cytometry ( Figure S61 ) and their AQP3 cellular distribution by immunocytofluorescence, respectively ( Figure 1 ). As expected from the literature and mRNA levels [ 23 ], the breast cancer cell line MCF-7 highly express AQP3. Ovarian cancer cells SKOV-3 also showed marked AQP3 expression, followed by the lung cancer cells A549 and the skin malignant melanoma cells A375. Regarding cellular localization, in the case of the melanoma cells A375, AQP3 was found to be also localized in the nuclear membrane ( Figure 2 ) as evidenced by fluorescence microscopy. The antiproliferative activities of the complexes are summarized in Table 1 . Overall, the compounds show very moderate anticancer effects in all cell lines, with compounds C1 and C2 being the least toxic (EC 50 > 80 μM). Compounds C3 and C4 were moderately cytotoxic, but their scarce stability in the aqueous environment may affect their antiproliferative effects. Compounds C10 – C12 were the most effective in the series, particularly against the melanoma A375 cells. It is worth mentioning that these three compounds are the only neutral ones, and may display different uptake and accumulation in cancer cells with respect to the cationic derivatives. Of note, the ligands L1 – L12 generally showed reduced antiproliferative effects with respect to the corresponding gold complexes ( Table S1 ). 3. Materials and Methods 3.1. General Information All reagents and solvents used have been obtained from Haereus (Hanau, Germany), Sigma Aldrich (St. Louis, MO, United States), Fluorochem (Old Glossop, UK), Alfa Aesar (Ward Hill, MA, USA) or Acros (Loughborough, UK) and were used as received unless specified. The identity and purity (≥95%) of the complexes were unambiguously established using high-resolution mass spectrometry and multinuclear NMR spectroscopy. 1 H, 13 C and 19 F NMR spectra were recorded on a Bruker Avance II300, II400 or II500 spectrometers (Bruker, Coventry, UK) at room temperature (r.t.) and referenced internally to residual solvent peaks [ 24 ]. The coupling constants are reported in Hertz. HR-ESI-MS spectra were obtained in acetonitrile/methanol on a Thermo Finnigan LCQ DecaXPPlus quadrupole ion-trap instrument (Thermo Fisher Scientific, Paisley, UK) operated in positive ion mode over a mass range of m / z 150–2000. IR spectra were measured on a Shimadzu IRAffinity-1S FT-IR (ATR) (Shimadzu, Milton Keynes, UK). Ligand functionalization reactions were monitored by thin-layer chromatography (Merck 60 F254 silica gel). Column chromatography was carried out manually using silica gel (Fluorochem; 40–63 μm, 60 Å) or on a Biotage Isolera automated flash purification system (ZIP cartridge, 5–10 g). The absorption and emission spectra of the ligands and corresponding complexes were recorded on Cary 5000 or 60 UV–Visible NIR (Agilent, Wokingham, UK), Cary Eclipse Fluorescence spectrophotometers (Agilent). 3.2. Compounds Synthesis 3.2.1. General Procedure for the Synthesis of Ligands L1 – L9 Pyridylbenzimidazole ligand L10 is commercially available. Functionalised pyridylbenzimidazole derivatives L1 [ 21 ], L4 [ 25 ], L7 [ 22 ], L8 [ 22 ], L9 [ 22 ], L11 [ 25 ], and L12 [ 18 ] have been synthetized following protocols reported in the literature. Ligands L2 and L3 have been obtained adapting protocols used to produce L1 and L4 , and have also been already reported in the literature [ 26 ]. The purity of the compounds was confirmed by elemental analysis, and all of them showed purity greater than 98%. Compounds L5 and L6 have been obtained using the following general protocol: A solution of 1 eq. of pyridylbenzimidazole (488 mg, 2.50 mmol) and 1.5 eq. of K 2 CO 3 (518 mg, 3.75 mmol) in 5 mL of DMF was stirred at room temperature for 30 min. Then, 1.2 eq. (3.00 mmol) of methyl-4-(bromomethyl)benzoate (in the case of L5 ; 687 mg) or 4-(trifluoromethyl)benzyl bromide (in the case of L6 ; 717 mg) were added to the mixture and left to stir at room temperature for approximately 4 h (the reaction was followed by TLC using a 1:1 ethyl acetate/hexane eluent system). The solution was then concentrated and water was added. The product was extracted several times by ethyl acetate. Organic layers were combined, washed with water and dried over MgSO 4 . After filtration and evaporation of the solvent, the crude products L5 and L6 were obtained and purified by column chromatography either manually using a 1:1 mixture of ethyl acetate and hexane or on an automated flash purification system using a gradient of ethyl acetate in hexane. L5 . White powder, 83% isolated yield (712 mg, 2.07 mmol). R f (1:1 AcOEt/hexane) ≈ 0.6. NMR 1 H (CDCl 3 ): 8.56 (ddd, J = 7.9, 4.0 and 2.7 Hz, 1H, H a ), 8.44 (dt, J = 8.0 and 1.0 Hz, 1H, H d ), 7.92–7.87 (m, 3H, 2xC H –Ph + H e ), 7.81 (td, J = 8.0 and 1.8 Hz, 1H, H c ), 7.33–7.25 (m, 5H, H h + H b + H f + H g + H e ), 7.20 (d, J = 8.5 Hz, 2H, 2xC H –Ph), 6.14 (s, 2H, C H 2 ), 3.77 (s, 3H, C H 3 ). NMR 13 C{ 1 H} (CDCl 3 ): 166.8 ( C O 2 Me), 150.4 ( C IV ) , 149.9 ( C IV ), 148.7 ( C a ), 142.8 ( C IV ), 142.8 ( C IV ), 137.0 ( C c ), 136.8 ( C IV ), 130.0 (2x CH –Ph), 129.3 ( C IV ), 126.7 (2x CH –Ph), 124.7 ( C d ), 124.0, 123.9, 123.0 ( C b , C f , C g ), 120.3 ( C e ), 110.5 ( C h ), 52.2 ( C H 3 ), 48.9 ( C H 2 ). ESI-TOF-MS (positive mode) for C 21 H 18 N 3 O 2 ([M + H] + ): calc. 344.1399, exp. 344.1404 (err. 1.5 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 313 (14,123). IR (ATR): ν (cm −1 ) 1709 (ν C–H bending ), 1428 (ν C–H stretching ), 1276 (ν C–O stretching ), 1110 (ν C–N stretching ), 845 (ν C=C bending ). Anal. Calcd for C 21 H 17 N 3 O 2 (343.39): C, 73.45; H, 4.99; N, 12.24. Found: C, 73.48; H, 4.97; N, 12.20. L6 . White powder, 88% isolated yield (774 mg, 2.19 mmol). R f (1:1 AcOEt/hexane) ≈ 0.6. NMR 1 H (CDCl 3 ): 8.55 (m, 1H, H a ), 8.44 (d, J = 8.0 Hz, 1H, H d ), 7.84 (d, J = 7.8 Hz, 1H, H e ), 7.79 (td, J = 7.8 and 1.8 Hz, 1H, H c ), 7.46 (d, 2H, 2xC H –Ph), 7.47–7.22 (m, 6H, 2xC H –Ph + H h + H b + H f + H g ), 6.20 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (CDCl 3 ): 150.4 ( C IV ) , 149.8 ( C IV ), 148.7 ( C a ), 142.8 ( C IV ), 141.7 ( C IV ), 137.1 ( C c ), 136.7 ( C IV ), 129.7 (q, C IV –CF 3 , 2 J C – F = 32.0 Hz), 126.8 (2x CH –Ph), 125.7 (q, 2x C H–C–CF 3 , 3 J C – F = 3.8 Hz), 124.7 ( C d ), 124.2 (q, C IV F 3 , 1 J C – F = 270 Hz), 124.1, 123.9, 123.2 ( C b , C f , C g ), 120.4 ( C e ), 110.4 ( C h ), 48.7 ( C H 2 ). NMR 19 F{ 1 H} (CDCl 3 ): −62.5 (s, C F 3 ). ESI-TOF-MS (positive mode) for C 20 H 15 N 3 F 3 ([M + H] + ): calc. 354.1218, exp. 354.1225 (err. 2.0 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 313 (23,130). IR (ATR): ν (cm −1 ) 1444 (ν C–H stretching ), 1328, 1157 (ν C–F stretching ), 1107 (ν C–N stretching ), 833 (ν C=C bending ). Anal. Calcd for C 20 H 14 N 3 F 3 (353.11): C, 67.98; H, 3.99; N, 11.89. Found: C, 67.95; H, 3.93; N, 11.82. 3.2.2. General Procedure for the Synthesis of the Cationic Complexes C1 – C9 Complex C1 was previously reported by us [ 15 ], and the same procedure was used to obtain complexes C2 – C9 . Thus, to a solution of 1 eq. of pyridylbenzimidazole ligand L1 – L9 in acetonitrile (2.5 mL) was added an aqueous solution (15 mL) of 1 eq. of NaAuCl 4 and 3 eq. of KPF 6 , and the resulting mixture was stirred 3 h at room temperature. The resulting yellow to orange precipitate was filtered, washed with water, ethanol and diethyl ether and dried under vacuum. C2. Yield: 68% (480 mg, 0.76 mmol). NMR 1 H (DMSO- d 6 ): 8.93 (m, 1H, H a ), 8.29 (m, 1H, H d ), 8.21 (m, 1H, H c ), 8.05 (m, 1H, H e ), 7.89 (m, 1H, H h ), 7.76 (m, 1H, H b ), 7.61 (m, 2H, H f + H g ), 4.87 (m, 2H, C H 2 ), 1.50 (s, 3H, C H 3 ). NMR 13 C{ 1 H} (DMSO- C3. Yield: 89% (561 mg, 0.78 mmol). NMR 1 H (DMSO- d 6 ): 8.92 (m, 1H, H a ), 8.29–8.19 (m, 2H, H d + H c ), 8.08 (m, 1H, H e ), 7.90 (m, 1H, H h ), 7.78 (m, 1H, H b ), 7.66–7.58 (m, 2H, H f + H g ), 4.85 (m, 2H, C H 2 ), 1.85 (m, 2H, C H 2 ), 1.17 (m, 10H, C H 2 ), 0.82 (m, 3H, C H 3 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 150.3 ( C a ), 146.9 ( C IV ), 143.9 ( C IV ), 138.8 ( C c ), 136.9 ( C IV ), 133.6 ( C IV ), 127.5 ( C g ), 127.1 ( C f ), 126.7 ( C d ), 126.6 ( C b ), 115.7 ( C h ), 113.8 ( C e ), 46.3 ( C H 2 ), 31.6 ( C H 2 ), 29.5 ( C H 2 ), 28.9 ( C H 2 ), 28.8 ( C H 2 ), 26.2 ( C H 2 ), 22.5 ( C H 2 ), 14.4 ( C H 3 ). ESI-TOF-MS (positive mode) for C 20 H 25 N 3 Cl 2 Au ([M + ]): calc. 574.1091, exp. 574.1100 (err. 1.6 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 312 (16,006). IR (ATR): ν (cm −1 ) 2927 (ν C–H stretching ), 1488, 1474 (ν C–H stretching ), 1039 (ν C–N stretching ), 831, 746 (ν C=C bending ). C4. Yield: 99% (403 mg, 1.61 mmol). NMR 1 H (DMSO- d 6 ): 8.83 (m, 1H, H a ), 8.30 (m, 1H, H d ), 8.17 (m, 1H, H c ), 7.87 (m, 1H, H e ), 7.78 (m, 1H, H h ), 7.70 (m, 1H, H b ), 7.55–7.49 (m, 2H, H f + H g ), 7.29–7.25 (m, 5H, 5xC H –Ph), 6.21 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 149.9 ( C a ), 147.2 ( C IV ), 144.5 ( C IV ), 138.1 ( C c ), 135.5 ( C IV ), 133.7 ( C IV ), 128.6 ( C H–Ph), 127.7 ( C H–Ph), 126.9 ( C b ), 126.5 ( C d ), 125.7 ( C f ), 125.7 ( C g ), 116.1 ( C e ), 113.0 ( C h ), 48.7 ( C H 2 ). ESI-TOF-MS (positive mode) for C 19 H 15 N 3 Cl 2 Au ([M + ]): calc. 552.0309, exp. 552.0307 (err. −0.4 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 313 (21,527). IR (ATR): ν (cm −1 ) 1497, 1475, 1454 (ν C–H stretching ), 831, 746 (ν C=C bending ). C5. Yield 48% (265 mg, 0.351 mmol). NMR 1 H (DMSO- d 6 ): 8.80 (m, 1H, H a ), 8.31 (m, 1H, H d ), 8.17 (t, J = 5.7 Hz, 1H, H c ), 7.92–7.88 (m, 3H, 2xC H –Ph + H e ), 7.77 (m, 1H, H h ), 7.69 (m, 1H, H b ), 7.58–7.50 (m, 2H, H f + H g ), 7.41–7.39 (m, 2H, 2xC H –Ph), 6.30 (s, 2H, C H 2 ), 3.80 (s, 3H, C H 3 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 165.9 ( C O 2 Me), 149.9 ( C a ), 145.0 ( C IV ), 141.3 ( C IV ), 138.3 ( C c ), 134.5 ( C IV ), 129.5 ( C H–Ph), 129.1 ( C IV ), 127.3 ( C H–Ph), 126.6 ( C b ), 125.9 ( C f+g ), 125.7 ( C d ), 116.5 ( C e ), 112.9 ( C h ), 52.2 ( C H 3 ), 48.8 ( C H 2 ). ESI-TOF-MS (positive mode) for C 21 H 17 N 3 O 2 Cl 2 Au ([M + ]): calc. 610.0363, exp. 610.0350 (err. −2.1 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 312 (23,448). IR (ATR): ν (cm −1 ) 1706 (ν C–H bending ), 1496, 1481 (ν C–H stretching ), 1285, 1018 (ν C–O stretching ), 1108 (ν C–N stretching ), 845 (ν C=C bending ). C6. Yield: 99% (435 mg, 0.568 mmol). NMR 1 H (DMSO- d 6 ): 8.81 (d, J = 4.3 Hz, 1H, H a ), 8.32 (d, J = 7.8 Hz, 1H, H d ), 8.18 (td, J = 7.8 and 1.3 Hz, 1H, H c ), 7.92 (d, 1H, J = 7.7 Hz, H e ), 7.80 (d, 1H, J = 7.8 Hz, H h ), 7.72–7.68 (m, 3H, H b + 2xC H –Ph), 7.59–7.53 (m, 2H, H f + H g ), 7.52–7.49 (m, 2H, 2xC H –Ph), 6.31 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 150.2 ( C a ), 147.3 ( C IV ), 143.9 ( C IV ), 140.3 ( C IV ), 138.5 ( C c ), 133.6 ( C IV ), 132.9 ( C IV ), 128.2 (q, C IV –CF 3 , 2 J C–F = 31.5 Hz), 127.9 (2x CH –Ph), 127.0 ( C b ), 126.6 ( C f ), 126.4 ( C g ), 126.0 ( C d ), 125.6 (q, 2x C H–C–CF 3 , 3 J C–F = 3.9 Hz), 123.8 (q, C IV F 3 , 1 J C–F = 272 Hz), 115.9 ( C e ), 113.2 ( C h ), 48.8 ( C H 2 ). NMR 19 F{ 1 H} (DMSO- d 6 ): −61.0 (s, C F 3 ), −70.0 (d, P F 6 , 1 J P–F = 712.4 Hz). ESI-TOF-MS (positive mode) for C 20 H 14 N 3 Cl 2 F 3 Au ([M + ]): calc. 620.0182, exp. 620.0212 (err. 4.8 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 312 (24,699). IR (ATR): ν (cm −1 ) 1481 (ν C–H stretching ), 1324, 1121, 1113 (ν C–F stretching ), 1068 (ν C–N stretching ), 834 (ν C=C bending ). Anal. Calcd for C 20 H 14 N 3 Cl 2 AuPF 9 (766.18): C, 31.35; H, 1.84; N, 5.48. Found: C, 31.35; H, 1.88; N, 5.48. C7. Yield: 87% (312 mg, 0.436 mmol). NMR 1 H (DMSO- d 6 ): 8.82 (d, J = 4.2 Hz, 1H, H a ), 8.31 (d, J = 8.0 Hz, 1H, H d ), 8.15 (t, J = 7.6 Hz, 1H, H c ), 7.86 (d, 1H, J = 7.6 Hz, H e ), 7.78 (d, 1H, J = 7.1 Hz, H h ), 7.88 (t, 1H, J = 5.3 Hz, H b ), 7.50 (m, 2H, H f + H g ), 7.35–7.32 (m, 2H, 2xC H –Ph), 7.15–7.11 (m, 2H, 2xC H –Ph), 6.19 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 162.9 ( C IV ) , 160.5 ( C IV ) , 150.0 ( C a ), 147.4 ( C IV ) , 144.6 ( C IV ) , 138.3 ( C c ), 133.9 ( C IV ), 133.0 (d, C IV F, 1 J C–F = 203 Hz), 129.4 (d, 2x CH –Ph, 3 J C–F = 8.4 Hz), 126.7 ( C b ), 126.0 ( C f ), 126.0 ( C g ), 125.9 ( C d ), 116.3 ( C e ), 115.6 (d, 2x CH –Ph, 2 J C–F = 21.5 Hz), 113.1 ( C h ), 48.2 ( C H 2 ). NMR 19 F{ 1 H} (DMSO- d 6 ): −114.3 (s, Ph– F ), −70.0 (d, P F 6 , 1 J P–F = 711.9 Hz). ESI-TOF-MS (positive mode) for C 19 H 14 N 3 Cl 2 FAu ([M + ]): calc. 570.0214, exp. 570.0236 (err. 3.9 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 312 (25,542). IR (ATR): ν (cm −1 ) 1460 (ν C–H stretching ), 1232 (ν C–F stretching ), 835 (ν C=C bending ). Anal. Calcd for C 18 H 12 N 3 O 2 Cl 2 AuPF 7 (702.14): C, 31.86; H, 1.97; N, 5.87. Found: C, 31.82; H, 2.00; N, 5.88. C8. Yield: 99% (394 mg, 0.500 mmol). NMR 1 H (DMSO- d 6 ): 8.76 (d, J = 4.4 Hz, 1H, H a ), 8.31 (d, J = 7.9 Hz, 1H, H d ), 8.14 (td, J = 7.8 and 1.6 Hz, 1H, H c ), 7.90–7.86 (m, 2H, H h + H e ), 7.66 (m, 1H, H b ), 7.58–7.51 (m, 2H, H f + H g ), 6.36 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 149.3 ( C a ), 148.5 ( C IV ) , 146.7 ( C IV ) , 146.0 (dd, C IV F, 1 J C–F = 246 Hz), 138.3 ( C c ), 137.1 ( C IV ), 135.2 ( C IV ), 126.0 ( C b ), 125.4 ( C g ), 125.3 ( C f ), 125.0 ( C d ), 117.9 ( C e ), 112.1 ( C h ), 110.9 (t, C IV –Ph, 2 J C–F = 17 Hz), 39.9 ( C H 2 ). NMR 19 F{ 1 H} (DMSO- d 6 ): −141.9 (Ph– F ), −154.3 (Ph– F ), −162.4 (Ph– F ), −70.0 (d, P F 6 , 1 J P–F = 711.9 Hz). ESI-TOF-MS (positive mode) for C 19 H 10 N 3 Cl 2 F 5 Au ([M + ]): calc. 641.9837, exp. 641.9874 (err. 5.8 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 311 (23,441). IR (ATR): ν (cm −1 ) 1474 (ν C–H stretching ), 1337, 1128 (ν C–F stretching ), 1029 (ν C–N stretching ), 838 (ν C=C bending ). C9. Yield: 99% (443 mg, 0.603 mmol). NMR 1 H (DMSO- d 6 ): 8.82 (d, J = 4.2 Hz, 1H, H a ), 8.29 (d, J = 8.0 Hz, 1H, H d ), 8.20 (t, J = 7.6 Hz, 1H, H c ), 7.91 (d, 1H, J = 7.6 Hz, H e ), 7.78 (d, 1H, J = 7.1 Hz, H h ), 7.88 (t, 1H, J = 5.3 Hz, H b ), 7.57 (m, 2H, H f + H g ), 7.19–7.09 (m, 4H, 4xC H –Ph), 6.20 (s, 2H, C H 2 ). NMR 13 C{ 1 H} (DMSO- d 6 ): 162.6 (d, C IV F, 1 J C–F = 247 Hz), 162.4 (d, C IV F, 1 J C–F = 247 Hz), 150.2 ( C a ), 147.2 ( C IV ) , 143.6 ( C IV ), 139.9 (t, C IV Ph, 3 J C–F = 9.6 Hz), 138.5 ( C c ), 133.4 ( C IV ), 132.4 ( C IV ), 127.1 ( C b ), 126.7, 126.5 ( C f , C g ), 126.1 ( C d ), 115.7 ( C e ), 113.2 ( C h ), 110.6 (d, 2x CH –Ph, 2 J C–F = 26.1 Hz), 103.5 (t, C H–Ph, 2 J C–F = 25.8 Hz), 48.5 ( C H 2 ). NMR 19 F{ 1 H} (DMSO- d 6 ): −109.1 (s, 2xPh– F ), −70.0 (d, P F 6 , 1 J P–F = 712.6 Hz). ESI-TOF-MS (positive mode) for C 19 H 13 N 3 Cl 2 F 2 Au ([M + ]): calc. 588.0120, exp. 588.0150 (err. 5.1 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 313 (14,367). IR (ATR): ν (cm −1 ) 1442 (ν C–H stretching ), 1332, 1091 (ν C–F stretching ), 1039 (ν C–N stretching ), 833, 792 (ν C=C bending ). 3.2.3. General Procedure for the Synthesis of the Neutral Complexes C10 – C12 The synthesis of complex C10 was already reported by us [ 15 ] and others [ 19 ], and was adapted to the synthesis of C11 and C12 . To a solution of 1 eq. of pyridylbenzimidazole ligand L10 – L12 in acetonitrile (1 mL) was added an aqueous solution of 1 eq. of KOH in water (6 mL). The reaction mixture was stirred at room temperature for 15 min. An aqueous solution of 1 eq. of NaAuCl 4 (6 mL) was then added, and the resulting mixture was stirred overnight at room temperature. The resulting dark brown precipitate was filtered; washed with water, ethanol and diethyl ether; and dried under vacuum. C11. Yield: 57% (100 mg, 0.18 mmol). NMR 1 H (500 MHz, DMSO- d 6 ): 9.11–8.90 (m, 3H, 3xC H ), 8.78 (m, 1H, C H ), 8.42 (m, 2H, 2xC H ), 8.21 (m, 1H, C H ), 8.13 (m, 1H, C H ), 7.86–7.74 (m, 3H, 3xC H ), 7.67 (m, 1H, C H ). NMR 13 C{ 1 H} (125.77 MHz, DMSO- d 6 ): 160.8 ( C IV ), 150.3 ( C H), 145.4 ( C IV ), 145.0 ( C IV ), 138.0 ( C H), 134.9 ( C IV ), 128.8 ( C H), 127.6 ( C H), 127.4 ( C H), 125.5 ( C H), 124.6 ( C H), 124.5 ( C H), 123.4 ( C H), 122.7 ( C H), 122.5 ( C H), 121.0 ( C H), 120.4 ( C H). ESI-TOF-MS (positive mode) for C 20 H 12 N 3 ClAu ([M – Cl + ]): calc. 526.0385, exp. 526.0386 (err. 0.2 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 333 (20,891), 362 (16,528). IR (ATR): ν (cm −1 ) 1428, 1452 (ν C–H stretching ), 1034 (ν C –N stretching ), 774 (ν C=C bending ). C12. Yield: 47% (87 mg, 0.15 mmol). NMR 1 H (500 MHz, MeCN- d 3 ): 8.63–8.47 (m, C H ), 8.35 (m, C H ), 8.27 (m, C H ), 8.23 (m, C H ), 8.14–7.98 (m, C H ), 7.59–7.57 (m, C H ). NMR 13 C{ 1 H} (125.77 MHz, DMSO- d 6 ): 161.2 ( C IV ), 150.4 ( C H), 149.6 ( C H), 145.7 ( C IV ), 145.5 ( C IV ), 138.0 ( C H), 135.6 ( C IV ), 131.7 ( C IV ), 128.3 ( C H), 127.8 ( C H), 127.2 ( C H), 126.6 ( C H), 125.0 ( C H), 124.5 ( C IV ), 123.4 ( C H), 122.8 ( C IV ), 122.6 ( C IV ), 122.2 ( C IV ), 120.0 ( C H), 119.7 ( C H), 119.5 ( C IV ), 118.3 ( C H). ESI-TOF-MS (positive mode) for C 22 H 12 N 3 ClAu ([M − Cl + ]): calc. 550.0385, exp. 550.0393 (err. 1.5 ppm). UV–Vis (DMSO): λ max (nm) (ε, cm −1 ·mol −1 ·dm 3 ) 338 (28,904), 353 (28,264), 366 (19,892), 388 (20,031). IR (ATR): ν (cm −1 ) 1442 (ν C–H stretching ), 1064 (ν C–N stretching ), 738 (ν C=C bending ). 3.3. UV–Visible and Fluorescence Spectroscopy The data are reported as the absorption maximum wavelength (λ max , in nm) and corresponding molar extinction coefficient at λ max (ε, in L·mol −1 ·cm −1 ). Macro quartz cuvettes with a path length of 1 cm were used. The sample concentrations were chosen to obtain a maximum absorbance of around 0.8 to then measure the quantum yields. Relative quantum yields of fluorescence of the samples were obtained by comparing the areas under the corrected emission spectra with a standard absorbing in the same region than the sample. Measurements were performed in degassed DMSO (Sigma-Aldrich, spectroscopic grade ≥99.9%) at 298 K. Quinine sulfate (Φ F = 0.546 in 0.5 M H 2 SO 4 , λ ex = 366 nm) was used as the standard [ 26 ]. In all Φ F calculations, the correction for the solvent refractive index (η) was applied: DMSO: η = 1.479; H 2 O (or H 2 SO 4 ): η = 1.333 [ 27 ]. The following equation was used to calculate the quantum yield of the sample (Φ F, x ), in which Φ F, St is the reported quantum yield of the standard, F is the integral photon flux, f is the absorption factor, and η is the refractive index of the solvent used. The x subscript denotes the sample, and St denotes the standard, and the fluorescence spectra between the sample and the standard were recorded at the same λ ex [ 28 ]: ϕ F , x = ϕ F , S t . F x F S t . 3.4. X-ray Diffraction Analysis Complexes C6 and C7 were allowed to form crystals by slow diffusion of pentane into a corresponding complex’s concentrated solution in a mixture of acetonitrile and dicholoromethane at room temperature. The crystals were analysed at the UK National Crystallography Service in Southampton [ 29 ]. A suitable yellow block-shaped crystal from C6 (0.050 × 0.040 × 0.030) mm 3 and from C7 (0.080 × 0.050 × 0.020) mm 3 , were selected and mounted on a MITIGEN holder in perfluoroether oil and mounted on a Rigaku 007HF diffractometer equipped with Varimax confocal mirrors and an AFC11 goniometer and HyPix 6000HE detector. The crystals were kept at T = 100.00(10) K during data collection. Using Olex2 [ 30 ], the structures were solved with the ShelXT [ 31 ] structure solution program, using the Intrinsic Phasing solution method. The models were refined with version 2014/7 of ShelXL [ 32 ] using Least Squares minimisation, and the ORTEP views are presented in Figures S62 and S63 . (CCDC 1868645 and 1868642 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected] )). 3.5. UV–Visible Absorption Spectroscopy The new Au(III) complexes were tested for their stability in aqueous media (1× Phosphate Buffer Saline (PBS, Corning), pH 7.4) in the presence or absence of 2 eq. GSH (vs. 1 eq. of Au(III) complex). The solutions were prepared from a stock solution of the Au(III) complex at a concentration of 10 −2 M in DMSO and diluted to reach a concentration of 10 −4 M in PBS. The absorption spectra were recorded over time at room temperature using a Cary 500 UV–Visible NIR spectrophotometer. 3.6. Aquaporins Inhibition Venous blood samples were obtained from healthy human volunteers following a protocol approved by the Ethics Committee of the Faculty of Pharmacy of the University of Lisbon (Instituto Português de Sangue Protocol SN-22/05/2007). Informed written consent was obtained from all participants. Blood samples, collected in citrate anticoagulant (2.7% citric acid, 4.5% trisodium citrate and 2% glucose), were centrifuged at 750× g for 5 min at 4 °C. Plasma and buffy coat were discarded. Pelleted erythrocytes were washed three times in PBS (KCl 2.7 mM, KH 2 PO 4 1.76 mM, Na 2 HPO 4 10.1 mM, NaCl 137 mM, pH 7.4), diluted to 0.5% haematocrit and immediately used for experiments. hRBC mean volume in isotonic solution was determined using a CASY-1 Cell Counter (Schärfe System GmbH, Reutlingen, Germany) and was calculated as 82 fL. Stopped-flow experiments were performed on a HI-TECH Scientific PQ/SF-53 apparatus, with 2 ms dead time, temperature controlled and were interfaced with a microcomputer. Measurements of water permeability (Pf) and glycerol permeability (Pgly) were performed as described in References [ 12 , 33 ]. Briefly, 100 µL of the suspension of fresh erythrocytes (0.5%) was mixed with an equal volume of hyperosmotic PBS containing 200 mM sucrose (a non-permeable osmolyte that induces water outflow and subsequent cell shrinkage) and 200 mM glycerol (a permeable osmolyte that induces first fast cell shrinkage due to water outflow and then glycerol influx in response to its chemical gradient, followed by water influx with subsequent cell reswelling). The kinetics of cell volume change were measured from the time course of 90° scattered light intensity at 400 nm until a stable light scatter signal was attained. For each experimental condition, 5–7 replicates were analysed. Pf was estimated by Pf = k (Vo/A)(1/Vw(osm out )∞), where Vw is the molar volume of water, Vo/A is the initial cell volume to area ratio, (osm out )∞ is the final medium osmolarity after the applied osmotic gradient, and k is the single exponential time constant fitted to the light scattering signal of erythrocyte shrinkage. Pgly was estimated by Pgly = k (Vo/A), where k is the single exponential time constant fitted to the light scattering signal of glycerol influx in erythrocytes. In inhibition experiments, cells were incubated with different concentrations of complexes from freshly prepared stock solutions, for 30 min at r.t. before stopped-flow experiments. A time-dependent inhibition assay for all the tested compounds over several hours of incubation showed no further increase of inhibition after 30 min at r.t. The inhibitor concentration necessary to achieve 50% inhibition (IC 50 ) was calculated by nonlinear regression of the dose-response curves (Graph Pad Prism, Inc, San Diego, CA, USA) to the equation: y = y min + ( y max − y min )/(1 + 10((LogIC 50 − Log[Inh])H)), where y is the percentage inhibition obtained for each concentration of inhibitor [Inh], and H is the Hill slope. All solution osmolarities were determined from freezing point depression on a semi-micro osmometer (Knauer GmbH, Berlin, Germany) using standards of 100 and 400 mOsM. 3.7. Cell Lines and Culture Conditions Human cell lines of lung adenocarcinoma (A549), breast carcinoma (MCF-7), ovarian adenocarcinoma (SKOV-3) and skin malignant melanoma (A375) were obtained from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 4.5 g/L glucose, Corning, Thermo Fisher Scientific), supplemented with 10% foetal bovine serum (One-Shot FBS, EU-approved South American Origin, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco). All cell lines were cultured at 37 °C, in a humidified atmosphere of 5% CO 2 and passaged when reaching confluence. 3.8. Immunocytofluorescence Round glass coverslips (Ø 13mm, VWR) sterilized by UV-light exposure were inserted in 24-well tissue culture-treated plates (Corning) prior to the addition of cells. Cells were seeded at a concentration of 125,000 cells/mL and incubated at 37 °C under humidified atmosphere with 5% CO 2 , for 24 h. Glass coverslips were removed from the wells and washed three times with 1× PBS (Gibco). Afterwards, cells were fixed for 20 min with 4% formaldehyde (Alfa Aesar) and permeabilized for 5 min with 0.2% Triton X-100 (Alfa Aesar) at room temperature (r.t.). Glass coverslips were then incubated with 1:500 primary anti-AQP3 antibody (rabbit anti-human, PA1488 BosterBio, Pleasanton, CA, USA) in 1× PBS with 5% normal human serum (NHS, Invitrogen, UK), for 1 h at r.t., followed by incubation with 1:500 secondary goat anti-rabbit Alexa Fluor ® 488 (ab150077, Abcam, Cambridge, MA, US), in 1× PBS with 5% NHS for 1 h, at r.t. and in the dark. Before/after each step, cells were washed thrice with 1× PBS. After removing excess PBS, cells were mounted on glass microscope slides (VWR) using Mowiol ® 4–88 (Sigma-Aldrich). Images were acquired on a Zeiss Axio Vert.A1 microscope and processed using Fiji (ImageJ) [ 34 ]. 3.9. Flow Cytometry Analysis For flow cytometry evaluation of AQP3 expression, samples of each cell line studied were prepared with 200,000 cells/sample. Cells were initially washed twice with 1× PBS (Gibco) and subsequently fixed with 4% formaldehyde (Alfa Aesar) for 30 min at room temperature (r.t.). Afterwards, cells were incubated with 1:500 dilution of anti-AQP3 antibody (rabbit anti-human, PA1488 Boster), in 1× PBS with 5% normal human serum (NHS, Invitrogen) and 0.1% Triton X-100 (Alfa Aesar) at r.t., for 1 h. Staining with primary antibody was followed by incubation with 1:500 secondary goat anti-rabbit Alexa Fluor ® 488 (ab150077, Abcam), in 1× PBS with 5% NHS and 0.1% Triton X-100 also for 1 h and at r.t., in the dark. Cells were kept on ice and away from direct light until analysed using a BD FACS Verse Flow Cytometer. Results were analysed using FlowJo 10.5.0. Firstly, samples were gated for live cells. Afterwards, stained and unstained samples were compared, in order to gate the positive population and the mean fluorescence, intensity (geometric mean) was taken from each positive sample peak. Data were normalized for the sample with the lowest AQP3 expression and results are shown as mean ± SEM of three independent experiments. 3.10. Antiproliferative Activities For evaluation of cell growth inhibition, cells were seeded in 96-well plates (Corning) at a concentration of 15,000 cells/well, grown for 24 h in 200 μL complete medium. Solutions of the samples with the desired concentration (1 to 100 µM) were prepared by diluting a freshly prepared stock solution (10 −2 M in DMSO) of the corresponding compound in aqueous DMEM medium, accordingly. Auphen (stock solution 10 mM in DMSO) was used as the reference compound. A negative control (medium only) was run for all the assays. After 24 h incubation, 200 μL of the compounds’ dilutions were added to each well, and cells were incubated for an additional 72 h. Afterwards, medium was removed and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Fluorochem) in 10× PBS (Corning) was added to the cells, at a final concentration of 0.3 mg/mL, and incubated for 3–4 h. After, the MTT solution was discarded and replaced with DMSO, to allow the formed violet formazan crystals to dissolve. The optical density was quantified in quadruplicates for each experiment, at 550 nm using a multi-well plate reader (VICTOR X, Perking Elmer, UK). The percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. The EC 50 values were calculated, using GraphPad Prism software, as the concentration showed 50% decrease in cell viability, compared to controls, using a nonlinear fit of concentration vs . response. Data is presented as mean ±SEM of at least three independent experiments. 4. Conclusions In conclusion, we have synthesized a new series of Au(III) compounds, cationic and neutral, featuring a pyridine-benzimidazole scaffold, which has been characterized via different methods. Varying the ligands’ substitution patterns influences the stability of the resulting Au(III) complexes in aqueous environment, as demonstrated by UV–visible spectroscopy. In general, all the compounds promptly react with the reducing agent GSH, except C11 – C12 . All the new gold-based complexes are potent inhibitors of human water and glycerol channel aqualgyceoroporin-3 (AQP3), while they are inactive as inhibitors of the water channel AQP1, as evidenced by stopped-flow spectroscopy in hRBC. While most of the compounds are scarcely active as antiproliferative agents against human cancer cells, the neutral complexes C10 – C12 showed promising anticancer activities, particularly in the melanoma A375 cancer cell line. Interestingly, while all the selected cell lines express AQP3, the melanoma cells display protein expression also at the level of the nuclear membrane, as shown by immunocytofluorescence. Thus, it may be hypothesized that C10 – C12 targets mainly AQP3 in the nuclear membrane after being taken up by the cancer cells. Overall, our results hold promise for the design of novel selective AQPs inhibitors to be used anticancer agents or as chemical probes to study the function of these interesting membrane channels. Supplementary Materials The following are available online at https://www.mdpi.com/2304-6740/6/4/123/s1 , CIF and checkCIF files. Scheme S1: Synthetic pathways to ligands L1 – L12 . Figure S1: 1 H NMR (400.13 MHz, CDCl 3 ) spectrum of ligand L5 . Figure S2: 13 C{ 1 H} NMR (100.61 MHz, CDCl 3 ) spectrum of ligand L5 . Figure S3: 1 H NMR (400.14 MHz, CDCl 3 ) spectrum of ligand L6 . Figure S4: 13 C{1H} NMR (100.61 MHz, CDCl 3 ) spectrum of ligand L6 . Figure S5: 1 H NMR (500.13 MHz, DMSO- d 6 ) spectrum of complex C2 . Figure S6: 13 C{ 1 H} NMR (125.77 MHz, DMSO- d 6 ) spectrum of complex C2 . Figure S7: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C3 . Figure S8: 13 C{ 1 H} NMR (125.77 MHz, DMSO- d 6 ) spectrum of complex C3 . Figure S9: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C4 . Figure S10: 13 C{ 1 H} NMR (125.77 MHz, DMSO- d 6 ) spectrum of complex C4 . Figure S11: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C5 . Figure S12: 13 C{ 1 H} NMR (100.61 MHz, DMSO- d 6 ) spectrum of complex C5 . Figure S13: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C6 . Figure S14: 13 C{ 1 H} NMR (100.61 MHz, DMSO- d 6 ) spectrum of complex C6 . Figure S15: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C7 . Figure S16: 13 C{ 1 H} NMR (100.61 MHz, DMSO- d 6 ) spectrum of complex C7 . Figure S17: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C8 . Figure S18: 13 C{ 1 H} NMR (100.61 MHz, DMSO- d 6 ) spectrum of complex C8 . Figure S19: 1 H NMR (400.13 MHz, DMSO- d 6 ) spectrum of complex C9 . Figure S20: 13 C{ 1 H} NMR (100.61 MHz, DMSO- d 6 ) spectrum of complex C9 . Figure S21: 1 H NMR (500.17 MHz, DMSO- d 6 ) spectrum of complex C11 . Figure S22: 13 C{ 1 H} NMR (125.77 MHz, DMSO- d 6 ) spectrum of complex C11 . Figure S23: 1 H NMR (500.17 MHz, MeCN- d 3 ) spectrum of complex C12 . Figure S24: 13 C{ 1 H} NMR (125.77 MHz, DMSO- d 6 ) spectrum of complex C12 . Figure S25: Absorption and emission spectra of ligand L1 . Figure S26: Absorption and emission spectra of complex C1 . Figure S27: Absorption and emission spectra of ligand L2 . Figure S28: Absorption and emission spectra of complex C2 . Figure S29: Absorption and emission spectra of ligand L3 . Figure S30: Absorption and emission spectra of complex C3 . Figure S31: Absorption and emission spectra of ligand L4 . Figure S32: Absorption and emission spectra of complex C4 . Figure S33: Absorption and emission spectra of ligand L5 . Figure S34: Absorption and emission spectra of complex C5 . Figure S35: Absorption and emission spectra of ligand L6 . Figure S36: Absorption and emission spectra of complex C6 . Figure S37: Absorption and emission spectra of ligand L7 . Figure S38: Absorption and emission spectra of complex C7 . Figure S39: Absorption and emission spectra of ligand L8 . Figure S40: Absorption and emission spectra of complex C8 . Figure S41: Absorption and emission spectra of ligand L9 . Figure S42: Absorption and emission spectra of complex C9 . Figure S43: Absorption and emission spectra of ligand L10 . Figure S44: Absorption and emission spectra of complex C10 . Figure S45: Absorption and emission spectra of ligand L11 . Figure S46: Absorption and emission spectra of complex C11 . Figure S47: Absorption and emission spectra of ligand L12 . Figure S48: Absorption and emission spectra of complex C12 . Figure S49: UV–Visible spectra of the Au(III) complex C1 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C1 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S50: UV–Visible spectra of the Au(III) complex C2 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C2 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S51: UV–Visible spectra of the Au(III) complex C3 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C3 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S52: UV–Visible spectra of the Au(III) complex C4 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C4 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S53: UV–Visible spectra of the Au(III) complex C5 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C5 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S54: UV–Visible spectra of the Au(III) complex C 6 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C6 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S55: UV–Visible spectra of the Au(III) complex C7 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C7 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S56: UV–Visible spectra of the Au(III) complex C8 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C8 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S57: UV–Visible spectra of the Au(III) complex C9 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C9 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S58: UV–Visible spectra of the Au(III) complex C10 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C10 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S59: UV–Visible spectra of the Au(III) complex C11 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C11 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S60: UV–Visible spectra of the Au(III) complex C12 (10 −4 M) in PBS (pH 7.4) recorded over time (left); and of C12 before and after addition of GSH (2 eq.) recorded over time at room temperature (right). Figure S61: Normalized mean fluorescence intensity (MFI) of AQP3, detected using a secondary Alexa Fluor®488-labelled antibody. Results were normalized for the sample with the lowest expression (A375) and results are expressed as mean ±SEM of three independent experiments. * p < 0.03. Figure S62: X-ray structure of crystals of C6 . Thermal ellipsoids drawn at the 50% probability level. Figure S63: X-ray structure of crystals of C7 . Thermal ellipsoids drawn at the 50% probability level. Table S1: Antiproliferative activities (EC 50 values) of ligands in human SKOV-3, A375, MCF-7, and A549 cells after 72 h incubation. Author Contributions Conceptualization, A.C. and G.S.; methodology, M.N.W., A.C., A.d.A., A.M., G.S.; formal analysis, A.M., A.d.A., B.A.; investigation, B.A., M.N.W., A.d.A., A.M.; resources, A.C. and G.S.; data curation, M.N.W., B.A., A.d.A., A.M.; X-ray data acquisition and analysis, W.T.K. and S.J.C; writing—original draft preparation, A.C., M.N.W.; writing—review and editing, all authors; visualization, B.A., A.d.A., A.M.; supervision, A.C., G.S.; funding acquisition, A.C., G.S. Funding This research received no external funding. Acknowledgments Cardiff University and Fundação para a Ciência e Tecnologia, Portugal (project PTDC/BTM-SAL/28977/2017 and PhD fellowship to A.M. 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Representative IC 50 curve for the inhibition of glycerol permeation via AQP3 by the Au(III) complexes C6 ( A ) and C11 ( B ) in hRBC after 30 min incubation. Figure 2. Human AQP3 expression and localization in human cancer cells by immunocytofluorescence. Human AQP3 expression (green) in A549, A375, SKOV-3 and MCF-7 cancer cells, with nuclei stained with DAPI (blue). Scale bars represent 25 μm. Figure 2. Human AQP3 expression and localization in human cancer cells by immunocytofluorescence. Human AQP3 expression (green) in A549, A375, SKOV-3 and MCF-7 cancer cells, with nuclei stained with DAPI (blue). Scale bars represent 25 μm. Table 1. AQP3 inhibitory effects (IC 50 values) measured in hRBC after 30 min incubation; and antiproliferative activities (EC 50 values) of Au(III) compounds in human SKOV-3, A375, MCF-7, and A549 cells after 72 h incubation, measured by the MTT assay. Table 1. AQP3 inhibitory effects (IC 50 values) measured in hRBC after 30 min incubation; and antiproliferative activities (EC 50 values) of Au(III) compounds in human SKOV-3, A375, MCF-7, and A549 cells after 72 h incubation, measured by the MTT assay. Compound AQP3 Inhibition EC 50 [μM] 1 IC 50 [μM] 1 SKOV-3 A375 MCF7 A549 Auphen 0.80 ± 0.08 7.00 ± 2.00 1.7 ± 0.3 3.00 ± 0.05 1.07 ± 0.09 C1 1.018 ± 0.137 >80 >80 >80 >80 C2 0.881 ± 0.015 >80 >80 >80 >80 C3 1.825 ± 0.017 ( n = 2) 41 ± 13 23 ± 1 40 ± 4 57 ± 2 C4 0.85 ± 0.21 56 ± 12 69 ± 3 63 ± 1 81 ± 9 C5 0.80 ± 0.10 >50 >50 25 ( n = 1) >50) C6 0.69 ± 0.06 >50 34 ( n = 1) 38 ( n = 1) 47 ( n = 2) C7 n.d. n.d. n.d. n.d. n.d. C8 n.d. n.d. n.d. n.d. n.d. C9 0.72 ± 0.05 >50 >50 >50 >50 C10 >50 17 ± 7 5 ± 2 12 ± 1 >50 C11 0.82 ± 0.13 ( n = 2) 33 ± 5 12 ± 2 29 ± 8 >50 C12 n.d. 41 ±13 13 ± 2 17 ± 3 45 ± 3 1 Values represented as mean (±SEM) of at least three independent experiments ( n ), unless otherwise stated. n.d. = non determined.	https://www.mdpi.com/2304-6740/6/4/123/htm
(PDF) Poverty-Health Nexus: The Case of Pakistan PDF \| On Mar 5, 2011, Abo Ul and others published Poverty-Health Nexus: The Case of Pakistan \| Find, read and cite all the research you need on ResearchGate Poverty-Health Nexus: The Case of Pakistan Figures Descriptive Statistics … Correlation Matrix … Estimates of the Model -1 … Estimates of the Model-2 … Estimates of the Model-2 (Continued). … Figures - uploaded by Imran Chaudhry Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations Middle Eastern Finance and Economics ISSN: 1450-2889 Issue 11 (2011) © EuroJournals Publishing, Inc. 2011 http://www.eurojournals.com/MEFE.htm Poverty-Health Nexus: The Case of Pakistan Abo ul Hassan Ph.D Research Fellow, Department of Economics Bahauddin Zakariya University, Multan, Pakistan E-mail: adam_smith_17@hotmail.com Shahnawaz Malik Professor of Economics, Bahauddin Zakariya University, Multan, Pakistan E-mail: shahnawazmalik@bzu.edu.pk Imran Sharif Chaudhry Associate Professor of Economics. Bahauddin Zakariya University, Multan, Pakistan E-mail: imranchaudhry@bzu.edu.pk Sadia Khalil M.Sc. (Economics) Bahauddin Zakariya University, Multan, Pakistan E-mail: sad_ia4i@hotmail.com Abstract This paper investigates t he effects of health status prox y by l ife expectancy and the expenditures on health along with the foremost socio-economic factors on the poverty levels proxy by head coun t ratio in Pakistan. Although it is a well document ed fact that the health indicators in Pakistan have been increas ed remarkably since its independence, yet its status is still far behind amo ng the nations of the world. Again one can easily analyze the contradiction between the soaring rate of fundin g in health sector and its outcomes. The dilemma a simple question whether the expenditures on health alone for the provision of health services to the masses is sufficient enough to alleviate poverty in Pakistan, or rather, if t here are other socio-economic factors which are c rucial to alleviate poverty in Pakistan so far. Th e paper had applied the methods of l east squares to investigate the relationship between poverty and health status in Pakistan. The stu dy constructs strong r elationship between health and poverty in Pakistan. The paper asserts that health is an important determinant of economic per formance and there exists a strong relationship between poverty and measures o f he alth. It has also argued that the key improvements in health would cause hefty economic gains. Thus, the idea is verified that impro ved health status increases the human skill and earning capacity which eventuall y reduces the poverty levels. In the end th e paper suggests the suitable policies which can improve the health status for a greater cause of poverty reduction. Keywords: Life expectancy, health, literacy, poverty Middle Eastern Finance and Economics - Issue 11 (2011) 143 I. Introduction The Millennium Development Goals driv es the attention towards poverty alleviation for which health has been assumed a key factor. These goals explicate the agenda of The World Health Organization while focusing on “Beyond its intrinsic value to individuals, h ealth is also central to ov erall human development and to the reduction of poverty”. The growing importance of good health has been recently receipted b y the international commu nity to tackle t he incidence of poverty. Poor health is not only a cause but also a consequence of pove rty. The direct relationship among health, poverty and economic growth is a well documented fact. However the situation is paradoxical in Pakistan. Muhammad Mushtaq khan (2006) has reviewed this confusing economic statement of Pakistan. He says that despite its better economic pe rformance and reasonable economic growth the coun try allocates minimal resour ces for health development. The a verage share of the health sector in the national budget remains in decimals in most of the budgetary allocations. In addition to the low average share o f the health sector, Pakistan’s major health indicators like life expectancy are low and the related health indicators like infant mortality are high. If the si tuation goes on in a similar way, Pakistan will be left behind among all the nations of the world who are now focusing on the importance of health for development. After having the awareness of the emergence o f the need of time, we have tried to explore the following questions with poli cy implications. a) Does investing in health reduce poverty? b) How much other relevant variables contribute in poverty reduction? c) Either increasing health expenditures is more productive or provision of health services is bett er by improving life expectancy? II. A Brief Literature Review Health includes all s orts of health related activities, such as provision o f health services. In many developing countries health services are often ineffective, with the result that many among the ma ny poor do not hav e access to the public health services they need. If people have access to effective health services, it is an essential measure of social protection to pr event the spiral from ill health to poverty. Theodore w, Schultz (1961), one of the pioneers of t he human development approach diverted the world’s attention towards the importance of he alth and education in human development issues. He agrees that health activities act as the most powerful agent of improving both the quality and quantity of life th at the effe ctiveness of healt h activities ca nnot be viewed in isolation from rest of the planning and development efforts in the country. The role of human capital in accelerating economic development is well recognized in the literature. Thus for fosterin g economic development and pove rty reduction i n general (Romer, 1986; Lucas, 1988; Squire, 1993; Ravallion and Chen, 1997; Sen, 1999; and S chultz, 1999), better health enhances the effective and sustained use of the knowledge and skills that individuals acquire through education (Schultz; 1999). Barro (1 996) further argues that better health can reduce the depreciation of education capital, and thus increases the favorable effect of education on growth. As Bloom and Canning (2000; 2003) have shown, healthier individuals might affect the economy in four way s: (a) The y might be more productiv e at work and so earn higher incomes; (b) They may spend more time in the labor force, as less healthy people take sickness absence or retire early; (c) The y may invest more in their own education, which will increase their productivity; and (d) They may save more in expectation of a longer life. III. Data and Methodology Our present study used s econdary data analysis in order to unfold t he health connections with pov erty levels. Our data sets consist of 37 annual observati ons from 1973-2009 on the selected va riables. The poverty data sets are collected mainly from Malik (1988), Amjad and Kemal (1997), Jamal (2003) and various issues of Pakistan economic surve y up to 2010. The other vari ables have been found from the evaluation reports and surveys conducted b y Federal Bureau of Statistics. Due to non availability of the 144 Middle Eastern Finance and Economics - Issue 11 (2011) values of some variables for the year 2009 we have used estimated values by the Federal Bureau of Statistics Pakistan. Moreover we have completed our data set on absolute poverty (HCR) by applying the linear interpolation technique. We have also tried to keep in view the problem of endogeniety while selecting the explanatory v ariables for our anal ysis. We have tried our level best to incorporate alm ost all the important variables necessary for Povert y-Health N exus. In order to highlight the contribution of health in poverty alleviation, we have used health expenditures as percentage of GDP in our analysis. In addition to this, we have used life expectancy as a proxy for health status which may always be maint ained by the provision of health services. We have used this proxy for male and females to mention the gender gap present in our economy. We have employed the poverty regression models to explore the Poverty-Health Nexus. In our first model, growth rate, openness, consumer price index, health expendit ures as a p ercentage of GDP and the pr evious year’s head c ount ratio are all explanatory variables wi th head count ratio (HCR) as the dependent variable. In second mod el, we have used literac y rate and life ex pectancies for male and female and dropped consumer price index owing to its insignificant status. IV. Results and Discussions a) Descriptive Statistics Analysis Our data s et consists of 38 years of annual observations from 1972-2009 on the selected variabl es. We have used the log of head count ratio (HCR) of previous year as one of our explanatory variables. We have carried out all our econometric analysis including the descriptive statistics by using E-views which gives us 37 obs ervations after adjusting end points. Now we take sto ck of the measure of central tendency and dispersion to find out whether our sam ple is drawn from the normal population. The table 1 elaborat es that the average of head count ratio for the present study period is 28.01 percent with standard deviation of 6.177 percent. The average of growth rate is 5.19 percent with a standard deviation of 2.12 percent. The averages for the remaining variables (openness, consumer price index, health ex penditures as percentage of GDP, litera cy rate, life expectancy for males and life expectancy for fem ales) are 33.90 percent, 60.32 percent, 0.74 percent, 37.96 percent, 60.72 percent and 61.07 percent respe ctively with the values of st andard deviations 3.12, 43.76, 0.17, 11.51, 3.16 and 3.16 are given accordingly. On e important thing to be noted is that the variation in the l iteracy is much higher then the variation in the health variables and rest of th e i ndependent variables except consumer price index. Table 1: Descriptive Statistics HCR GROWTH OPEN CPI HEPGDP LITR LEXPM LEXPF Mean 28.01 5.19 33.90 60.32 0.74 37.96 60.72 61.07 Median 25.83 4.96 34.51 43.90 0.73 36.65 60.54 61.07 Maximum 45.75 10.22 38.91 149.20 1.19 57.00 66.21 66.86 Minimum 20.71 0.81 2 7.72 7.4 0 0.40 22.10 55.33 54.82 Std. Dev. 6.77 2.12 3.12 43.76 0.17 11 .51 3.16 3.61 Skewness 0.88 -0.04 -0.37 0.53 0 .53 0.19 0.06 -0.05 Kurtosis 2.90 2.69 2.28 1.87 3.62 1.54 1.89 1.75 Jarque-Bera 4.8 8 0 .17 1.72 3.84 2.38 3.60 1.96 2.49 Probability 0.09 0.92 0.42 0.15 0.30 0 .17 0 .38 0.29 Observations 38 38 38 38 38 38 3 8 38 Middle Eastern Finance and Economics - Issue 11 (2011) 145 Table 2: Correlation Matrix HCR GROWH OPEN CPI HEPGDP L ITR LEXPM LEXPF HCR 1.00 GROWTH -0. 29 1.00 OPEN -0.63 0.11 1.00 CPI -0.15 -0.10 0.20 1.00 HEPGDP -0.67 0.07 0.26 -0.22 1.00 LITR -0.22 -0.16 0.27 0 .98 -0.12 1.00 LEXPM -0.33 -0.06 0.28 0.97 -0.03 0.98 1. 00 LEXPF -0.35 -0.09 0.30 0 .96 -0.01 0.98 0.99 1.00 Now we discuss the Jerque-Bera test which is based on classical measures of skewness and kurtosis. We have measured the robustness of skewness and tail weight. As we know that symmetric distribution has zero skewness, we find that our distributio n is asymmetric. As far as the direction of the skewness of the variables is concerned, head count ratio (HCR), consumer price index, heal th expenditures as percentage of GDP, literacy rate, life expectancy for male are positively skewed whereas growth rate, openness, and life expectancy for female are negatively sloped. It can be seen from table 1 that all va riables are minutely skewed to the left or right. Kurtosis characterizes the relative peakedness or flatness of the distribution compared to normal distribu tion. The positive values of the kurtosis show that the variables are superguassian. The significant value of kurtosis of health expenditures as a percentage of GDP also verifies its characteristic of leptokurtic distribution that shows its higher and sharper central peak along with longer tails comp ared to a normal distribution whereas all the remaining variables are platykurtic as the values of fourth standardized moments lie below 3 but we can see the values of head count ratio (HCR) and growth rate are very close t o the desired value of kurtosis. The j oint venture of skewness and kurtosis determines whether a random variable follows a normal distribution or not? The Jerque-Bera test for the normality o f the concerned distributions suggests that the residuals for all the variables included in this study are normally distributed. Table 2 presents the strength of the relationship of variables under consideration. The degree of relationship of variables is also determined by correlation matrix given in table 2. All t he variables are inversely correlated with each other. The r esults depict a pictu re of the degree of correlation between the regressors and the regressand. Openness and health expenditures as a percentage of GDP are highl y correlated with the incidence of poverty whereas life expectanc y for m ale and female are moderately correlated and all other remaining variables are weakly correlated with the dependent variable. The Poverty Regression Model-1 In this section, we present the results of estimations of poverty-health mode l which we have developed after manipulations of including the most concerned variables and excluding the variables whi ch have weaker or no effect on our predictand. In order to find out the role of health in povert y connections, we have applied the regression techniques on our poverty-health model. The Model, Results and Interpretations LHCR= β о + β 1 LGROWTH + β 2 LOPEN + β 3 LCPI + β 4 LHEPGDP + β 5 LHCR (-1) + ε i Table 3 presents the estimation results of our poverty-health model in which log of head count ratio (HCR) is dependent variable with log of gross domestic product (GDP), log of openness, log of consumer price index (CPI), log of health expenditures as a percentage of gross domestic product and the log of previous year’s head count ratio; HCR (-1), all as regressors to run the model. The value of adjusted R-squared is 85 percent, implying that 85 percent of the variation i n the dependent v ariable is explained by the p redictors. The high value of R -squared throws light on the justification of our poverty-health model. Moreover, the value of V IF is 8.2 which is viewed as an evidence of the absence of multicollinearity. The value of Durbin h-statistics is 0.59 following the argument of no significant autocorrelation problem in the error. 146 Middle Eastern Finance and Economics - Issue 11 (2011) The coefficient for the l og of growth is having a ne gative sign which fulfills our theor etical expectations. The coefficient for the log of growth is highly si gnificant, indicating towards its dominant role in povert y alleviation. The results are also s upported by Michael Roemer and M ary K Gugerty (1997 ) who argues that growth in per capit a GDP can be and us ually are a powerful force in reducing poverty and Sarris (2001) who gives a strong support to the propos ition that overall economic growth reduces overall poverty. The coefficient of openness also shows an inverse relationship between the log of head count ratio and the log of openness. The coefficient for the log of openness is not found statistically significant. It has rather low effect on poverty or the minimum effect on regressand. The coefficient for the log of inflation is having a theoretically expected sign a nd implies a positive relationship on poverty. However the coefficient is not statistically significant. Our results also stay in line with th e findings of Romer and Romer (1998) and Agenor (1998) who have conclu ded that inflation also adds fuel to the fire of prevailing poverty levels. The co efficient for the log of health expenditures as a percentage of gross domestic product is highly si gnificant with the incidence of pover ty. The coefficient of our health variable is keeping a postulated negative sign and embellishes t he title of the research paper on being hi ghly significant and pervading immense effect on the poverty levels. Our results also second the findings of Mitra (2004) who observed that per capita income and public health expenditures posi tively influence health status, povert y declines with better health. In this way, our results are also supported by Straus and Thomas (1998) and Schul tz (1999) who emphasize that better health has a positive effect on the learning attitude and abilities of chil dren and leads to better educational outcomes and increases the efficiency of human capital formation by individuals and households. The coefficient for the log of previous year’s head count ratio is highly significant with poverty ratio. The results are supported by Ragner Nurkse who believes poverty itself as one of the major causes of poverty. Table 3: Estimates of the Model -1 Variable Coefficient Std. Error t-Statistic Prob. C 0.844911 0.483031 1.749188 0.0902 LGROWTH -0.075050 0.031040 -2.417840 0.0217 LOPEN -0.262549 0.213671 -1.228754 0.2284 LCPI 0.007776 0.021317 0.364773 0.7178 LHEPGDP -0.178023 0.096526 -1.844292 0.0747 LHCR(-1) 0.698887 0.128249 5.449460 0.0000 R-squared 0.874009 F-Statistic 43.00990 Adjusted R-squared 0.853688 Probability 0.000000 Durbin- h- statistic 0.59 The Poverty Regression Model- 2 It i s admit tedly realized that poverty remains in all the economies of the world in a great or small manner. We are trying to unfold the paradoxical status o f health in connection with poverty alleviation. The juggernauts of human development have recently realized the importa nce of health status to reduce the curse of poverty. The foremost task before us is to search a suitable proxy for health status. There are only two variables which could give a true picture of health status. The one is infant mortality and the other is life expectancy. Both the life and the death proxies encircle the overall health status independently. We have selected life expectancy at birth as a prox y variable to reflect the health status. We have postponed the idea of incorporating infant m ortality in our analysis as the data collected so far is unreliable due to some missing values. We have also included the gender analysis with reference to the health status in our model. The model is given below. Poverty = + β 1 LGROWTH+ β 2 LOPEN+ β 3 LLITR+ β 4 LLEXPM+ β 5 LLEXPF + β 6 LHCR (-1) + µ i Middle Eastern Finance and Economics - Issue 11 (2011) 147 Table 4 presents the estimation results for the poverty regression analysis. The depe ndent variable is the poverty head count ratio (HCR) and log of growth, log of openness, log of literacy, log of li fe expectanc y male, log of life expectancy fe male and the log of previous year’s he ad count ratio are all dependent variables in our econometric analysis. The ratio of adjusted R-squared is 85.9% which implies that approximately 85.9% variation in the depe ndent variable is explained by the independent variables elucidating the robustness of the results. Moreover the value of VIF is 7.1 indicating the absence of multicollinearit y. The value of The coefficient of LGROWTH is keeping a postulated negative sign implying the inverse relationship with the incidence of povert y. The coefficient for LGROWTH is highly significant statistically like the prev ious model. Our present results strengthen our faith t hat increases in growth decreases the poverty levels. The results again show a meager role of globalization, having its insignificant coefficient in the poverty regression model. The c oefficient of literacy rate is statistically significant with a ne gative sign, implying an inverse relationship with the incidence of poverty. According to Naushin Mehmood (the chief of research, PIDE), it has been esti mated that Pakistan could have attained an increase in per capita GDP , if it increased its literacy and primary school enro llment. Therefore, it c an be argued that literacy plays its role quite significantly in pove rty alleviation, which confirms our estim ation results. Our estimation results also match with the findings of T.W.Schultz (1961), Gary Becker (1964), Jacob Mincer (1958) and George Psacharopoulos (2002) who have examined the significant role of human capital in economic growth, giving the key importance to education. The coefficient for lo g of life expectanc y for males has an expected negative sign implying that an increase in the life expectancy for males caus es a reduction in the incidence 0f poverty. Although the coefficient is not stat istically significant even though it verifies the truth that in a male dominant society like Pakistan where majorit y of the financial responsibilities are held by males when increase is found in their life ex pectancies, helps to reduce povert y. Our results match with the findings of Bloom and Sachs (1998), Gallup and Sachs (2001), WHO (2001), and Bloom and Canning (2005) who believe that improving health can h ave equall y large indirect pay offs through accelerating economic growth. On the contrary, the results are found opposite for females to that of an increase in the life expectancies implying that an increas e in the life expectancy for females enhances the incidence of poverty. The logic is very clear that majority of the female side in Pakistan spend a dependent life and increase in their life expectancy means more burden on the ec onomy. The results are supported by Dr. I shrat Hussain who claims that gender disparities are quite significant and low parti cipation in labour forc e is preventing the country from reaching its p eak capacity. Moreov er according to the labour force survey 2008, Pakistan’s female labour force participation is still four times lo wer than the male labour f orce participation rate of 82.4 percent and an increase in the life expectancy of such a big dep endent side will just give rise to t he poverty levels. Poverty itself bein g a major reason of povert y is confirm ed by the coefficient for the log of previous year’s head count ratio. Th e coefficient is statistically significant with having a positive sign. Table 4: Estimates of the Model-2 Variable Coefficient Std. Error t-Statistic Prob. C -5.133816 2.580415 -1.989531 0.0558 LGROWTH -0.090225 0.032802 -2.750634 0.0100 LOPEN -0.050535 0.201244 -0.251115 0.8034 LLITR -0.2477 77 0.128522 -1.927898 0.0634 LLEXPM -0.026971 1.534981 -0.017571 0.9861 LLEXPF 3.421586 1.691907 2.022326 0.0521 148 Middle Eastern Finance and Economics - Issue 11 (2011) Table 4: Estimates of the Model-2 (Continued). LHCR(-1) 0.961261 0.089836 10.70022 0.0000 R-squared 0.8 82200 F-statistic 37.44466 Durbin-h statistic 1.4 V. Conclusions and Policy Implications In this paper, we focused the relationship between health and poverty which is the heart of the human development approach along with education. Health has both direct and i ndirect pa yoffs. People with good health may not only increase t heir personal incomes but also give rise t o the overall growth process and hence provi des reason for povert y reduction. In terms of polic y implications, it follows that in case of Pakistan, improving hea lth either by increasing the health e xpenditures or making arrangements to increase the life expectancy of males has an important and significant influence on the process of poverty alleviation. We have used life expectancy as a proxy variable to m easure the health status. Moreover, we have also checked the i mpact of health expenditure on poverty. We h ave used a very common, effective and popular variable: health exp enditures as a per centage of GDP to check the impact of public he alth expenditures on the incidence o f poverty. For the measurement of povert y, we have used absolute poverty in our analysis. We have estimated time series regression to check the impact of health variables alon g with other most important macro economic variables on the incidence of poverty. Our estimation results indicate that h ealth expenditures may pla y a key role in poverty alleviation as their impac t is significant on absolute povert y. The increase in life ex pectancy for males may reduce the poverty levels but its role in this contex t is meager. The reason lies behind the truth that the employment opportunities in Pakistan are narrowing day by day. Our paper also implicates that by increasing the employment opportunities both for male and female can make the increased life expectancy more effec tive and fruitful. As w e know health wins a prime importance in all the four approaches of human development, thereby supporting our estimation results. Our empi rical results indicate towards the importance of health issues i n fixi ng the curse of poverty. Growth and literac y also helps to reduce poverty whi ch is obvious from the r esults of our both poverty regression models. References [1] Agenor, Pierre-Richard ( 1998). Stabili zation policies, poverty and the Labour Market, Mimeo, IMF and World Bank. [2] Amjad, Rashid and Kemal, A.R. (1997). Macroeconomic policies and their impact on poverty alleviation in Pakistan. The Pakistan development review, 36(1), 39-68. [3] Barro (1996), “Health, Human Capital and Economic Growth”, Paper f or the Program on Public Policy and Health, Pan American Health Organization and World Health Organization (Washington: Pan American Health Organization). [4] Becker, Gary S. (1964). Human Capital. New York Colombia Universit y Press for NBER. [5] Bloom D. E, Canning, D (2000), The health and wealth of nations, Science 2000, 287, 1207- 1209. [6] Bloom, David E., and David Canning. 2005. “Health and Economic Gro wth: Reconciling the Micro and M acro Evidence.” Working Paper (February), Stanford University, Freeman Spogli Institute for International Economics, Palo Alto, CA. [7] Bloom, David E., and Jeffrey D. Sachs. 1998. “Geography, D emography, and Economi c Growth in Africa.” Brookings Papers on Economic Activity 2: 207–95. Middle Eastern Finance and Economics - Issue 11 (2011) 149 [8] Bloom, David, and David Canning (2003), “The Health and Poverty of N ations: From Theor y to Practice,” Journal of Human Development, Vol. 4, No. 1, pp. 47–71. [9] Gallup, John Luke, and Jeffrey D. Sachs. 2001. “The Economic Burden of Malaria.” American Journal of Tropical Medicine and Hygiene 64 (1 supplement, Januar y): 85–96. [10] Huss ain Dr. Ishrat (2010), Pakistan’s Econom ic Prospectus ; Challenge s and Opportu nities. A paper presented at the seminar on “Pakistan- China relations in 21 century” held at Beijing on March 1 8, 2010. [11] Jamal, H. (2003). Poverty and inequality during the adjustment dec ade: Empirical findings from household surveys. The Pakistan Development Review, 42(4), 125-136. [12] Khan Muhamm ad Mushtaq (2006), Health Polic y Analysis: The case of Pakistan ISBN-10:90- 5291-099-5, ISBN-13:978-90-5291-099-4, printed in the Neitherland b y Datawyse Maastricht. [13] Labour Force survey, Pakistan, 2008. [14] Lucas, Rober t E. Jr. (1988), “On the Mechanic of Economic Developm ent”, Journa l of Monetary Economic s, Vol. 22, pp. 3 –42 [15] Malik, Muhamm ad Hussain (1988). Some new Evidence on the incidence of poverty in Pakistan. The Pakistan Development Review, 27(4), 509-516. [16] Michael Roemer and M ary Kay Gugerty. "Does Economic Growth Reduce Poverty?" (Technical Paper). CAER II Discussion Paper No. 5, June 1997. [17] Mincer, Jacob (1958). Investment in Human Capital and Personal Income Distribution. Journal of Political Economy, 66. [18] Mitra, Arup (2004) What Motivates Transfer of Resources: Altruism or Pri nciple of Exchange? The Journal of Developing Areas - Volume 37, Number 2, spring 2004, pp . 31-48. [19] Noushin Mehmood , Research Repo rt#172 Educational Developments in Pakistan: Trends, Issues and Policy concerns. [20] Psacharopoulos, George and Patrinos, Harry A nthony (2002). “Returns to Investment: A Further Update. World Bank Policy Research Working Paper 2881, September, 2002. [21] Ravallion, Martin, and Shaohua Chen (1997), “What Can New Survey Data Tell Us About Recent Changes in Distribution and Poverty?”, World Bank Economic Review, Vol. 11, No. 2, pp. 357–82. [22] Romer, Christina and David Romer (1998). Mon etary P olicy and the Well-Being of the Poor. National Bureau of Economic Research Working paper 6793, November. [23] Romer, Paul (1986), “Increasing Returns and Long- Run Growth,” Journal of Political Economy, Vol. 94 , No. 5, pp. 1002 –37. [24] Sarris, Alexander H. (2001). The Role of Economic Development and Poverty Reduction: An Empirical and Conceptual Foundation. University of Athens, Athens. [25] Schultz, T. Paul (1999), “Health and Schoolin g Investments in Africa,” Journal of Economic Perspectives, Vol. 13, No. 3, pp. 67–88. [26] Schultz, T.W. (1961). Investment in Human Capital. The American Economic Review 1(2), 1- 17. [27] Schultz, Theodore W. (1961). Education and Economic Growth. In N.B. Henr y (ED). Social factor influencing education. Chicago: University of Chicago Press. [28] Sen, Amartya (1999), Development as Fredom. Oxford: Ox ford University Press. [29] Squire, Lyn (1993), “Fight ing Poverty,” Am erican Economic Review, Papers and Proceedings, Vol. 83, No. 2, pp. 377–82. [30] Strauss John; Thomas Duncan (1998), Health, Nutrition and Economic Development, Journal of Economic literature, vol: 36, no: 2. (June, 1998), PP.766-817. [31] WHO (2001), Macroeconomics and Health: Investing in Health for Economic Development, Geneva. Geography, Demography, and Economic Growth in Africa Some New Evidence on the Incidence of Poverty in Pakistan A number of studies have been done in the past to measure the level of poverty in Pakistan. These studies include Naseem (1973, 1977), Alauddin (1975), Mujahid (1979), Irfan and Amjad (1983), Kruijk and Leeuwen (1985) and Cheema (1985). The time periods covered by these studies are not the same. Moreover, in some cases the methodologies and results of these studies also differ. The present study covers the most recent data made available in the Household Income and Expenditure Survey (HIES) for 1984-85. Some selected previous Survey years have also been included in the study to see changes in poverty levels over time. The incidence of poverty is measured on the basis of both households and population. To determine the location of the poor, poverty levels have been estimated for rural and urban areas of the country. The Health and Wealth of Nations Empirical studies show that health improvements provide a significant boost to economic growth in developing countries. This leads to the view that health, like education, is a fundamental component of human capital, and suggests the notion of health-led growth. Better health leads to higher income, but there is also a positive feedback effect, giving rise to a beneficial situation where health and income improvements are mutually reinforcing. What Motivates Transfer of Resources: Altruism or Principle of Exchange? This paper focuses on the issue of remittances and other non-monetary transfers among individuals. Some of the recent studies gave a new dimension to the understanding of this issue by highlighting the aspect of self-interest and the principle of exchange, which may motivate the act of helping others rather than being guided entirely by altruism. Based on the sample survey in the low-income households of Delhi slums, this study collects evidence to reconfirm this viewpoint. The binomial logit framework used to model decisions of whether or not to remit and/or help others tends to favour the practice of reciprocation in explaining transfers of resources, after controlling for household size, education, duration of migration and contact at the place of origin of the migrants and income (savings). While household size tends to reduce the probability of transfer of monetary resources, other variables seem to raise the possibility of extending both monetary and non-monetary help. Informal networks based on exchange behaviour have important policy implications in the context of urban poverty reduction and slum management, as these informal practices can be used as inputs for designing programmes based on participatory approach, which would be more cost efficient than direct interventions. Schooling, health, and economic growth: reconciling the micro and macro evidence We compare the estimated effects of health in a macroeconomic production function model of economic growth with the effects that are found using calibration based on wage regressions. We allow for gradual adjustment in income level towards the steady state which means that both the level and growth of inputs can affect economic growth. We find that the estimated macroeconomic effects of health are positive, and not significantly different from the microeconomic estimates. We find similar results for education provided we instrument schooling levels with literacy rates to correct for measurement error. The Health and Poverty of Nations: From Theory to Practice Increasing Returns and Long Run Growth Health policy analysis: the case of Pakistan / Proefschrift Rijksuniversiteit Groningen. Met lit.opg. - Met samenvatting in het Nederlands.	https://www.researchgate.net/publication/349811595_Poverty-Health_Nexus_The_Case_of_Pakistan
JCM \| Free Full-Text \| A New Perspective for Potential Organ Damage Due to Iron-Mediated Oxidation in Thalassemia Major Patients Background: The aim of this study is to develop new perspectives to prevent or reduce potential organ damage due to iron-mediated oxidation in thalassemia major patients. Methods: Seventy patients were included in this study. Blood samples were taken from the patients before and after transfusion. Total thiol, native thiol, disulfide, disulfide/native thiol percentage ratio, ischemia modified albumin (IMA), total antioxidant status (TAS), total oxidant status (TOS), and ferroxidase levels were determined. Additionally, undepleted thiol level (UTL) was determined as a new parameter associated with organ damage. Results: After transfusion, the levels of native thiol, total thiol, disulfide, TAS, ferroxidase, and TOS were higher, while the IMA levels and disulfide/native thiol percent ratio were lower. Significant correlations were found between antioxidant and oxidant tests before and after transfusion. Additionally, a negative correlation was found between the TOS and UTL levels of the patients measured before the transfusion. Conclusion: In the present study, transfusion therapy increased both oxidation and the antioxidant levels. In addition, the term UTL has been introduced as a parameter that enables the determination of the oxidation level that may cause potential organ damage in transfusion-dependent thalassemia patients. Funda Eren 1,* , Ayça Koca Yozgat 2 , Esra Firat Oğuz 1 , Salim Neşelioğlu 3 , Rıdvan Firat 1 , Dilek Gürlek Gökçebay 2 , Hüsniye Neşe Yarali 2 , Namık Yaşar Özbek 2 and Özcan Erel 3 1 Department of Medical Biochemistry Laboratory, Ankara Bilkent City Hospital, 06800 Ankara, Turkey 2 Department of Pediatric Hematology and Oncology, Ankara Bilkent City Hospital, 06800 Ankara, Turkey 3 Department of Medical Biochemistry, Yıldırım Beyazıt University, 06010 Ankara, Turkey * Author to whom correspondence should be addressed. J. Clin. Med. 2023 , 12 (6), 2422; https://doi.org/10.3390/jcm12062422 Received: 9 February 2023 / Revised: 15 March 2023 / Accepted: 20 March 2023 / Published: 21 March 2023 (This article belongs to the Section Clinical Laboratory Medicine ) Browse Figure Abstract Background: The aim of this study is to develop new perspectives to prevent or reduce potential organ damage due to iron-mediated oxidation in thalassemia major patients. Methods: Seventy patients were included in this study. Blood samples were taken from the patients before and after transfusion. Total thiol, native thiol, disulfide, disulfide/native thiol percentage ratio, ischemia modified albumin (IMA), total antioxidant status (TAS), total oxidant status (TOS), and ferroxidase levels were determined. Additionally, undepleted thiol level (UTL) was determined as a new parameter associated with organ damage. Results: After transfusion, the levels of native thiol, total thiol, disulfide, TAS, ferroxidase, and TOS were higher, while the IMA levels and disulfide/native thiol percent ratio were lower. Significant correlations were found between antioxidant and oxidant tests before and after transfusion. Additionally, a negative correlation was found between the TOS and UTL levels of the patients measured before the transfusion. Conclusion: In the present study, transfusion therapy increased both oxidation and the antioxidant levels. In addition, the term UTL has been introduced as a parameter that enables the determination of the oxidation level that may cause potential organ damage in transfusion-dependent thalassemia patients. Keywords: antioxidant ; oxidant ; thalassemia ; transfusion 1. Introduction Thalassemias are hereditary blood disorders due to anomalies in the synthesis of the chains of hemoglobin [ 1 ]. ß-thalassemia (β-thalassemia) is a condition characterized by microcytic hypochromic anemia, an abnormal peripheral blood smear with reticulocytes, and decreased hemoglobin ß chain synthesis resulting in low amounts of hemoglobin A (HbA) [ 2 ]. Thalassemia is one of the most common inherited hemoglobinopathies and the prevalence is approximately 2% in Turkey [ 3 ]. Red cell suspension transfusion is the mainstay of therapy in patients with major thalassemia. A regular transfusion schedule and chelation therapy aimed at reducing iron overload allow for normal growth and development and can improve overall prognosis [ 1 , 4 ]. Intracellular enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and some other components such as vitamin E and reduced glutathione (thiol groups) provide protection from oxidative stress [ 5 ]. Overdose oxidative stress on cells can cause the oxidation of proteins, lipids, and DNA, leading to cell death and organ damage. Oxidative stress is believed to exacerbate the symptoms of many diseases, including hemolytic anemias. There are several studies showing oxidative stress in the pathogenesis of hemoglobinopathies such as ß-thalassemia, glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, and congenital dyserythropoietic anemias [ 6 , 7 , 8 ]. Irreversible organ damage occurs in many organs as a result of oxidative reactions occurring due to excess iron in patients with major thalassemia [ 9 ]. Although iron accumulation is tried to be prevented by chelation therapy, the main problem is organ damage caused by oxidation. It is very important to determine quantitatively and evaluate the oxidation level together with chelation therapy in transfusion-dependent thalassemia patients. The parameters selected in this study were chosen according to their ability to evaluate the presence of antioxidants and oxidants in the serum both cumulatively and in detail. Thiol groups are organic compounds that are at the forefront of antioxidant molecules and contain a sulfhydryl (-SH) group in their structure. It is suggested that oxidative stress leads to macromolecular damage and disruption of thiol redox circuits leading to abnormal cell signaling and dysfunctional redox control [ 2 , 10 ]. Most of the plasma thiol pool is derived from proteins, and some from molecules such as low molecular weight cysteine, cysteinylglycine, glutathione, homocysteine, and gama glutamylcysteine. When thiol groups in serum are oxidized, they form disulfide bonds. Disulfide bonds are converted back to thiol groups by the action of antioxidants in the serum. This condition is reversible and is in balance under physiological conditions. However, in many diseases, this balance is disturbed [ 11 ]. Thiol–disulfide balance tests consist of native thiol, total thiol, disulfide, and disulfide/native thiol ratio parameters. Total thiol is expressed as the sum of disulfide and native thiol levels, while disulfide/native thiol percent ratio is expressed as the ratio of oxidant and antioxidant levels [ 10 ]. Undepleted thiol level (UTL) is a term used for the first time for thalassemia patients. UTL is the mathematical difference of serum native thiol values measured after and before transfusion (UTL = serum native thiol value measured after transfusion–serum native thiol value measured before transfusion). The difference arises depending on the oxidation state of the patient prior to transfusion. It suggests that UTL may be related to the elimination of oxidation products found in thalassemia patients by the antioxidant thiol groups in the erythrocyte suspension. Ischemia modified albumin (IMA) is a marker that has been used in the diagnosis of ischemic disease such as coronary artery disease in recent years. In the presence of ischemia, when the balance between the oxidant and antioxidant system is disturbed, albumin undergoes structural and functional modifications. Thus, the metal-binding ability of albumin is impaired and the level of IMA increases [ 2 ]. Total antioxidant status measurement test (TAS) and total oxidant status measurement test (TOS) provide cumulative information about antioxidant and oxidant status in the serum [ 12 , 13 ]. Ferroxidase, a copper-containing oxidase protein, has oxidase activities against polyamines, polyphenols, and inorganic iron ions. It is also critical in iron metabolism. The primary physiological role of ferroxidase is associated with redox reactions in the organism [ 14 , 15 ]. Catalyzing the oxidation of ferrous ions (Fe 2+ ) to the less reactive ferric state (Fe 3+ ), the form required for iron binding to apotransferrin, is associated with the antioxidant activity of ceruloplasmin. Ferrous iron ions are known to be an important catalysts of hydroxyl radical formation. Ferroxidase activity removes reactive iron ions from the plasma, thus preventing the stimulation of lipid peroxidation by iron ions [ 16 ]. Up to date, there are several studies about the relationship between oxidative stress and ß-thalassemia. To our knowledge, this is the first comprehensive study aiming to evaluate the oxidant and antioxidant status before and after transfusion by detecting serum total thiol, native thiol, disulfide, IMA, TAS, TOS, and ferroxidase levels. The aim of this study is to develop new perspectives to prevent or reduce organ damage due to iron-mediated oxidation in thalassemia major patients. 2. Materials and Methods This study was conducted at the pediatric hematology department and medical biochemistry laboratory of the hospital. Seventy patients who were diagnosed with ß-thalassemia major receiving regularly scheduled blood transfusions were included in this study. Transfusion treatments are started when the patients are 6–9 months old and are performed regularly once a month. All patients are given deferasirox (30–40 mg/kg/day) as an iron chelator. In our country, all blood components are manufactured and distributed by the Turkish Red Crescent. According to the transfusion program, the patients were transfused with ABO and subgroup compatible, pre-storage leukofiltered, and non-irradiated packed erythrocyte units in a volume of 15 cc/kg every 3–4 weeks. The RBC units were preserved with SAG-M (saline, adenine, glucose, mannitol) and stored for a maximum 7 days. Patients included those with the diagnosis of thalassemia intermedia or minor, endocrinological disorders, cardiovascular and infectious diseases, and cancer. Additionally, insufficient samples were excluded from our study. Informed consents were obtained from the parents of all subjects. Two blood specimens were collected from the patients before and 2 h after transfusion into serum gel separator tubes and centrifuged at 1500× g for 20 min. Sera were aliquoted and stored at −80 °C until the analysis. Total thiol and native thiol were measured with a novel automated spectrophotometric method. Disulfide and disulfide/native thiol percentage ratios are calculated using the measured parameters [ 10 ]. UTL is the mathematical difference of serum native thiol values measured after and before transfusion (UTL = serum native thiol value measured after transfusion–serum native thiol value measured before transfusion). IMA level was measured spectrophotometrically by the method based on the cobalt binding capacity principle of albumin defined by Bar-Or et al. [ 17 ]. Total antioxidant status (TAS) and total oxidant status (TOS) levels were measured also with an automated direct measurement method developed by Erel O [ 13 , 18 ]. For ferroxidase enzyme activity, ferric iron ions (Fe 3+ ) were measured kinetically in a spectrophotometer using acetohydroxamic acid [ 19 ]. Our study was approved by Ankara City Hospital Ethics Committee number E2-22-1437. The distribution of the data was evaluated by using Kolmogorov–Smirnov analysis. Descriptive analyses were expressed as mean ± standard deviation for normally distributed variables and as median (interquartile range) for non-normally distributed variables. Paired t -test was used to determine the significance level of the difference between groups for normally distributed data, and the Wilcoxon test was used for data that were non-normally distributed. In addition, Pearson correlation analysis was performed for normally distributed variables and Spearman correlation analysis was performed for non-normal distribution. Categorical variables were presented as numbers and percentages. All statistical calculations were made using SPSS (Statistical Package for Social Sciences) software (v.22; IBM, Armonk, NY, USA). p values less than 0.05 were considered statistically significant for all analyses. Figures were created using GraphPad Prism (Version 6.0; GraphPad Software Inc., La Jolla, CA, USA). 3. Results The study included 70 patients with the diagnosis of ß-thalassemia receiving regular transfusion therapy, 36 (51.4%) females and 34 (48.6%) males. The mean age of the patients was 8.83 ± 4.32 years. After transfusion native thiol, total thiol, disulfide, TAS, ferroxidase, and TOS levels were found to be significantly higher ( p < 0.001). IMA level and disulfide/native thiol percent ratio were found to be significantly lower after transfusion ( p < 0.001) ( Table 1 and Figure 1 ). Figure 1. Antioxidant and oxidant parameters before and after transfusion in thalassemia patients ( ± ) ( n = 70). Table 1. The values of antioxidant and oxidant parameters before and after transfusion in thalassemia patients. Significant correlations were found between antioxidant and oxidant tests before and after transfusion ( Table 2 and Table 3 ). Table 2. The correlation analysis of oxidant and antioxidant status before transfusion. Table 3. The correlation analysis of oxidant and antioxidant status after transfusion. There is a significant negative correlation between the TOS levels and the UTL levels of the patients measured before the transfusion (r = −0.436; p < 0.001). 4. Discussion Blood transfusions, ineffective erythropoiesis, and intravascular hemolysis result in iron overload in many body tissues of ß-thalassemia patients. Iron overload leads to increased ROS (reactive oxygen species) production and oxidative stress [ 20 ]. Overproduction of ROS can cause cellular toxicity and organ dysfunctions [ 21 ]. Antioxidants protect cells against oxidative stress damage by eliminating or preventing ROS production [ 22 ]. It has been hypothesized that changes in erythrocytes in ß-thalassemia are associated with a constant oxidative stress resulting from excessive intracellular precipitation of alpha-globin chains and release of free iron [ 23 , 24 ]. Iron overload in ß-thalassemia leads to an enhanced generation of ROS and results in oxidative stress in the body. For compensation, antioxidants in the body take role to protect erythrocytes from oxidative stress. In previous studies, it has been indicated that increased antioxidant status was probably due to the increase in oxidative stress in ß-thalassemia patients receiving blood transfusions and iron chelators [ 4 , 25 ]. In our study, we evaluated the oxidant and antioxidant status of ß-thalassemia patients before and after blood transfusion. According to our findings, the levels of the antioxidants (TAS, native and total thiol, and ferroxidase) were significantly higher and oppositely the levels of the oxidants (disulfide/native thiol ratio and IMA) were significantly lower after transfusion. Moreover, the TOS levels, which show the oxidative status, were significantly higher after transfusion. The antioxidant parameters showed statistically significant correlation with each other and also the oxidant parameters showed statistically significant correlation with each other. Moreover, antioxidant and oxidant parameters were negatively correlated. In the study, strong negative correlations were obtained between the levels of native thiol, TAS, and ferroxidase, which are important antioxidant parameters, and IMA levels, which is one of the oxidative stress indicators. These results show that the oxidant state is dominant in patients with ß-thalassemia before transfusion. These results indicate that the antioxidant–oxidant balance is impaired in patients with ß-thalassemia before transfusion. It was observed that the disulfide/native thiol ratio, which indicates the antioxidant–oxidant balance through thiol metabolism, was also impaired before transfusion. In addition, a significant positive correlation between disulfide/native thiol and IMA levels supports the existence of oxidative stress. Additionally, significant positive correlation values observed between the antioxidant parameters native thiol, TAS, and ferroxidase levels are important for the reliability of the measurements ( Table 2 and Table 3 ). It is known that oxidative stress increases in ß-thalassemia patients [ 26 ]. It is thought that the reason for the increase in post-transfusion antioxidant status in our patients receiving regular transfusion therapy is related to the erythrocyte suspension given. TAS shows the antioxidant status of the body [ 12 ]. According to some studies, total antioxidant status (TAS) was not different between ß-thalassemia patients and healthy controls [ 27 ]. In our study, TAS levels were significantly higher after transfusion comparing with before transfusion. This result indicates that the transfusion therapy contributes to the increased antioxidant status. TOS levels were also significantly elevated after transfusion in the present study. The elevated iron levels in ß-thalassemia patients, even after transfusion, may lead to the elevation of lipid peroxides and oxidants through Fenton reaction [ 27 ]. In previous studies, serum IMA levels of ß-thalassemia patients were found to be significantly higher than controls [ 15 , 28 ], considering the presence of oxidative stress, ROS production, hypoxia, and anemia in ß-thalassemia [ 29 , 30 ] elevated IMA levels are expected to be observed in these patients. In our study, we found high IMA levels before transfusion in ß-thalassemia patients, consistent with the literature. After transfusion, the IMA levels were significantly lower. Transfusion causes an increase in hemoglobin levels and a decrease in hypoxia and oxidative stress. The presence of lower IMA levels in patients after transfusion may be associated with these changes. Ceruloplasmin ferroxidase activity converts iron from the ferrous to the ferric form that prevents the formation of ROS and reduces oxidative damage through the Fenton reaction [ 15 , 31 ]. In a previous study, ferroxidase activity in thalassemia patients was found to be higher as compared to healthy controls [ 14 ]. The high ferroxidase activity observed in ß-thalassemia may be attributed to mechanisms related to iron overload. In our study, ferroxidase levels were significantly elevated after transfusion. Transfusion therapy increases the iron overload, resulting in increased ferroxidase levels to prevent the formation of ROS via the Fenton reaction by oxidation of Fe 2+ to Fe 3+ . Thiol and disulfide homeostasis plays an important role in the antioxidant protection and detoxification process [ 2 ]. Our study was carried out to examine the changes in thiol and disulfide homeostasis. Total and native thiol levels were found to be significantly higher after transfusion. Disulfide levels were also found to be higher after transfusion. Disulfide bounds are the products of the oxidation of the thiols. The increase in disulfide bounds is due to the increase in thiol groups after transfusion. The decrease in the ratio of disulfide/native thiol indicates the decreased oxidative stress associated with thiol metabolism after transfusion. In addition, considering the metal chelation feature of thiol groups [ 32 ], it is thought that increased thiol levels after transfusion may increase the effectiveness of treatment by contributing to iron chelation therapy as well as antioxidant activity [ 33 ]. In patients with transfusion-dependent thalassemia, erythrocyte transfusion is the most important step in the treatment. However, as a result of oxidative reactions that occur due to excessive iron accumulation resulting from regular blood transfusion, irreversible damage may occur in many organs [ 9 ]. In this study, the increase in TOS levels after transfusion indicates the presence of iron-mediated oxidative stress in line with the literature. In addition, it was observed that TAS and native thiol levels, which reflect the antioxidant status, increased after transfusion. Studies to date have only mentioned the increase in oxidative stress and ignored the increase in antioxidants that eliminates some of the oxidative stress and thus partially reduces organ damage. To our knowledge, this point of view has been reported for the first time in this study. In addition, we think that determining thiol levels in serum samples taken before and after transfusion and keeping the UTL value under control can reduce organ damage. One of the causes of organ damage in patients with thalassemia major is damage to macromolecules due to iron-mediated oxidation and resulting in cell death. Although iron accumulation is tried to be prevented by chelation therapy, the main problem is cell damage caused by oxidation. For this reason, quantitative determination of oxidation level together with chelation therapy in transfusion-dependent thalassemia patients would be beneficial. In this study, native thiol levels, which have antioxidant properties, were measured in serum samples taken from patients before and after transfusion. Native thiol levels increased after transfusion in thalassemia patients ( Table 1 , Figure 1 ). The difference depends on the oxidation state of the patient prior to transfusion. In the present study, the difference in thalassemia patients was named as the thiol level that remained undepleted (undepleted thiol level—UTL). It was shown that there is a significant negative correlation between UTL and serum TOS levels measured before transfusion (r = −0.436; p < 0.001). In other words, the patients with low oxidation levels before transfusion have high UTL values, or the patients with high oxidation levels before transfusion have low UTL values. Based on this, it can be predicted that keeping the UTL values of patients under control with thiol-containing drugs (e.g., alpha lipoic acid, n-acetyl cysteine) may help to limit oxidative damage that may cause cell damage. There are many studies showing that the use of alpha-lipoic acid in thalassemia major patients reduces cardiovascular risk factors [ 34 ], limits damage caused by iron-mediated oxidative stress [ 35 ], and has an excellent iron chelating property [ 33 ]. These positive effects of alpha-lipoic acid suggest that it may be a way to determine the damage observed in thalassemia patients by UTL measurement. The quantitative determination of oxidation by UTL measurement is a prediction that cell damage known to occur due to oxidation can be detected. One of the limitations of our study was the limited number of patients. Additionally, we could evaluate only a limited number of oxidant and antioxidant parameters. The evaluation could be performed at the time of the diagnosis of the patients and the first transfusion therapy. Moreover, more detailed evaluation of the patients with organ damage is needed Additionally, any parameter showing the cell damage directly could not be evaluated. In conclusion, transfusion therapy in major thalassemia patients not only increased the oxidation levels but also increased the antioxidant levels. In addition, the term “undepleted thiol level” has been introduced as a parameter that enables the determination of the oxidation levels that may cause cell damage in transfusion-dependent thalassemia patients. Thus, it was suggested that the UTL value could be presented as a quantitative value related to the prediction of cell damage. According to the data, we believe that the results may contribute to the effectiveness of transfusion used in the treatment of ß-thalassemia and to limit possible organ damage. 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[ Google Scholar ] [ CrossRef ][ Green Version ] Figure 1. Antioxidant and oxidant parameters before and after transfusion in thalassemia patients ( ± σ x ¯ ) ( n = 70). Table 1. The values of antioxidant and oxidant parameters before and after transfusion in thalassemia patients. Parameters Before Transfusion After Transfusion p -Values Native thiol (µmol/L) 378.0 ± 150.2 458.0 ± 105.6 <0.001 * Total thiol (µmol/L) 411.4 ± 152.1 495.2 ± 107.7 <0.001 * Disulfide (µmol/L) 16.7 ± 3.4 18.5 ± 4.2 =0.005 * Disulfide/Native thiol ratio (%) 5.1 ± 1.1 4.3 ± 0.7 =0.008 * TAS (mmol/L) 0.98 ± 0.05 1.04 ± 0.06 <0.001 * Ferroxidase (U/L) 611.8 ± 208.1 720.1 ± 176.9 <0.001 * IMA (ABSU) 0.76 (0.14) 0.73 (0.06) <0.001 TOS (µmol/L) 2.14 (0.89) 3.7 (2.15) <0.001 TAS—total antioxidant status; TOS—total oxidant status; IMA—ischemia modified albumin; ABSU—absorbance unit; indicates a significant statistical difference with p < 0.05; * used paired sample t -test; ** used Wilcoxon signed rank test. Table 2. The correlation analysis of oxidant and antioxidant status before transfusion. Parameters Ferroxidase TAS TOS IMA Native thiol r 0.704 0.492 0.438 −0.775 p <0.001 * <0.001 * <0.001 * <0.001 * Total thiol r 0.710 0.505 0.438 −0.785 p <0.001 * <0.001 * <0.001 * <0.001 * Disulfide r 0.256 0.328 0.099 −0.331 p 0.02 * 0.003 * 0.377 0.002 * Disulfide/Native thiol r −0.567 −0.376 −0.298 0.692 p <0.001 * <0.001 * 0.007 * <0.001 * Ferroxidase r 1 0.479 0.378 −0.659 p <0.001 * <0.001 * <0.001 * TAS r 0.479 1 0.186 −0.703 p <0.001 * 0.094 <0.001 * TOS r 0.378 0.186 1 −0.291 p <0.001 * 0.094 0.008 * IMA r −0.659 −0.703 −0.291 1 p <0.001 * <0.001 * 0.008 * * indicates a significant statistical difference with p < 0.05. r: correlation coefficient. Table 3. The correlation analysis of oxidant and antioxidant status after transfusion. Parameters Ferroxidase TAS TOS IMA Native thiol r 0.421 0.164 0.300 −0.679 p <0.001 * 0.140 0.006 * <0.001 * Total thiol r 0.419 0.174 0.319 −0.677 p <0.001 * 0.118 0.003 * <0.001 * Disulfide r 0.089 0.133 0.252 −0.132 p 0.429 0.235 0.022 * 0.238 Disulfide/Native thiol r −0.286 −0.139 0.007 0.547 p 0.009 * 0214 0.951 <0.001 * Ferroxidase r 1 0.106 0.260 −0.361 p 0.341 0.018 * 0.001 * TAS r 0.106 1 0.185 −0.140 p 0.341 0.096 0.209 TOS r 0.260 0.185 1 −0.158 p 0.018 * 0.096 0.155 IMA r −0.361 −0.140 −0.158 1 p 0.001 * 0.209 0.155 * indicates a significant statistical difference with p < 0.05. r: correlation coefficient. MDPI and ACS Style Eren, F.; Koca Yozgat, A.; Firat Oğuz, E.; Neşelioğlu, S.; Firat, R.; Gürlek Gökçebay, D.; Yarali, H.N.; Özbek, N.Y.; Erel, Ö. A New Perspective for Potential Organ Damage Due to Iron-Mediated Oxidation in Thalassemia Major Patients. J. Clin. Med. 2023, 12, 2422. https://doi.org/10.3390/jcm12062422 AMA Style Eren F, Koca Yozgat A, Firat Oğuz E, Neşelioğlu S, Firat R, Gürlek Gökçebay D, Yarali HN, Özbek NY, Erel Ö. A New Perspective for Potential Organ Damage Due to Iron-Mediated Oxidation in Thalassemia Major Patients. Journal of Clinical Medicine. 2023; 12(6):2422. https://doi.org/10.3390/jcm12062422 Chicago/Turabian Style Eren, Funda, Ayça Koca Yozgat, Esra Firat Oğuz, Salim Neşelioğlu, Rıdvan Firat, Dilek Gürlek Gökçebay, Hüsniye Neşe Yarali, Namık Yaşar Özbek, and Özcan Erel. 2023. "A New Perspective for Potential Organ Damage Due to Iron-Mediated Oxidation in Thalassemia Major Patients" Journal of Clinical Medicine12, no. 6: 2422. https://doi.org/10.3390/jcm12062422 Eren, F.; Koca Yozgat, A.; Firat Oğuz, E.; Neşelioğlu, S.; Firat, R.; Gürlek Gökçebay, D.; Yarali, H.N.; Özbek, N.Y.; Erel, Ö. A New Perspective for Potential Organ Damage Due to Iron-Mediated Oxidation in Thalassemia Major Patients. J. Clin. Med. 2023, 12, 2422. https://doi.org/10.3390/jcm12062422	https://www.mdpi.com/2077-0383/12/6/2422/xml
Investigation of the effect of Berkovich and Cube Corner indentations on the mechanical behavior of fused silica using molecular dynamics and finite element simulation \| Request PDF Request PDF \| Investigation of the effect of Berkovich and Cube Corner indentations on the mechanical behavior of fused silica using molecular dynamics and finite element simulation \| In this study, elastic modulus and hardness of fused silica were evaluated and then simulated by molecular dynamics (MD) and finite element... \| Find, read and cite all the research you need on ResearchGate Investigation of the effect of Berkovich and Cube Corner indentations on the mechanical behavior of fused silica using molecular dynamics and finite element simulation December 2021 Ceramics International 48(32–35) DOI: 10.1016/j.ceramint.2021.12.201 Authors: Abstract In this study, elastic modulus and hardness of fused silica were evaluated and then simulated by molecular dynamics (MD) and finite element analysis (FEA). The reduced modulus, hardness of the surfaces, and elastic modulus caused by the process along the depth of the indentation on the surfaces are evaluated by the nanoindentation method on the fused silica. The obtained results indicate that applying different indenters such as Berkovich and Cube-Corner reduces the amount and size of surface microcracks. The load-displacement of various indenters such as Berkovich and Cube-Corner with similar load at about 50 mN for the Berkovich shows a 400 nm displacement and a 1600 nm for the Cube-Corner indenter. In this study, the MD was used to describe the mechanical behavior of fused silica structures with atomic nanostructure as simulated at T0 = 300 K and P0 = 1 bar as the initial condition. The mechanical constants such as hardness (with Berkovich tip), elastic modulus (with Berkovich tip), and fracture toughness (with Cube Corner tip) of fused silica nanostructure can be calculated from this MD simulation in which the hardness of the atomic matrix is obtained at 8.84 GPa. ... Such relevancies require using a proper maximum indentation depth while studying the hardness of FRP composites [54]. Moreover, the researchers introduced 3D micromechanical modeling or molecular dynamics to obtain hardness and elastic modulus of the constituents of composite structures from indentation point of view [55] [56] [57]. It is evident that theoretical and numerical modeling can play a crucial role in predicting the behavior of different materials and structures [58,59]. ... Indentation, Finite Element Modeling and Artificial Neural Network Studies on Mechanical Behavior of GFRP Composites in an Acidic Environment In this study, the indentation tests are performed with various forces using the Vickers indenter to investigate the mechanical properties, including the elastic modulus, hardness, and the plasticity index of pure, and incorporated glass fiber reinforced polymer (GFRP) composites with nanosilica and nanoclay. To study the effect of adding different nanoparticles on reducing the mechanical properties of immersed specimens in acid for 0, 1 and 3 months, incorporated composite specimens with 3 wt percent (wt. %) of nanoparticles were fabricated using hand lay-up method. Accordingly, the reduction in mechanical properties and increase in plasticity index was attributed to the penetration of acid into composite specimens during the immersion period, which resulted in the failure of matrix and fiber following increased moisture penetration. Adding the nanoparticles especially nanoclay, has alleviated the trend of drop in mechanical properties. In fact, for the incorporated composite specimen with nanoclay, the hardness and elastic modulus of the immersed samples in acid for 0 and 3 months indicated a decrease of 10.55 and 7.88%, respectively on grounds of hydrophobic nature of nanoclay. Conversely, incorporating nanosilica increased the plasticity index leading to higher rate of degradation, which was even more than pure sample, yet the pure sample had the lowest mechanical properties after 3 months of immersion. In addition, Finite element modeling (FEM) and artificial neural network (ANN) were used to respectively predict the indentation behavior of fabricated composite specimens and study the effects of immersion time in an acidic environment. The effect of penetrated oxygen particles on combustion time of coated Al hydride nanoparticles in an oxygenated medium by applying molecular dynamics method via Lennard-Jones and reactive force-field potentials The novelty of using the AGM and FEM for solutions of partial differential and ordinary equations along a stretchable straight cylinder Near the suction/injection area, a scientific definition for laminar boundary layer flow and heat transfer of an incompressible viscous flow over a stretching cylinder is given. In the study, differential equations with partial derivatives are converted into dimensionless coupled equations using numerical and analytical methods of Akbari- Ganji and Finite Elements Methods. The goal of this first stage of research and research on this topic is to use simplified forms to simplify equations using derivatives of simplified forms; the analysis of the displacement of the heat flux and the velocity gradient will be done using the changes of the Prandtl number. Based on the results obtained on this issue, it is found that the suction process increments surface firmness and quality, whereas the injection decreases surface skin friction. Also, at the points where the water and oil are attached to the surface of the cylinder, the heat has reached its maximum value, and as the distance increases along the Y axis, the temperature decreases. The highest temperature gradient is observed for water fluid. This shows that the use of water fluid around the cylinder accelerates the process of heat transfer from the surface to the outside of the boundary layer. One of the differences between the use of oil and water fluid around the cylinder according to the 2D contours is the difference in the temperature gradient of the two fluids. So that the highest temperature gradient is observed for water fluid. Reinforcing cement with nanoparticles is a way to reduce porosity and enhance the initial strength of cement. In this work, the calcium phosphate cement is reinforced with magnesium ion nanoparticles using the molecular dynamics simulation method. This work investigates the mechanical and thermal properties of simulated samples. In this approach, the ultimate strength, Young's modulus, and thermal stability are studied. The effects of different initial temperature values and pressures will be researched next. As the initial temperature increased from 300 to 400 K, the ultimate strength and Young's modulus decreased from 0.879 and 0.171 MPa to 0.843 and 0.154 MPa. Also, the thermal stability reduced from 1321 to 1294 K. This material has more strength at lower than higher temperatures. With increasing temperature, the materials become softer and lose their strength. As the initial temperature in the atomic samples increases, the mobility intensifies, and as a result, the stability of the structures decreases. Increasing the initial pressure from 1 to 5 bar, the ultimate strength and Young's modulus enhanced from 0.879 and 0.171 MPa to 0.889 and 0.178 MPa. Also, the thermal stability increased from 1321 K to 1371 K by increasing the initial pressure to 5 bar. As the pressure increases, the amplitude of the particle oscillation decreases. The nanoparticle-reinforced cement matrix shows more appropriate behavior in high-pressure environments. It is expected that the addition of Mg nanoparticles will increase the strength of calcium phosphate cement and be used for use in bone lesions. The response of GFRP nanocomposites reinforced with functionalized SWCNT under low velocity impact: experimental and LS-DYNA simulation investigations	https://www.researchgate.net/publication/357234283_Investigation_of_the_effect_of_Berkovich_and_Cube_Corner_indentations_on_the_mechanical_behavior_of_fused_silica_using_molecular_dynamics_and_finite_element_simulation
Pfau v. Reed - Opposition \| OSG \| Department of Justice Docket number: No. 98-1846 Supreme Court Term: 1999 Term Court Level: Supreme Court No. 98-1846 In the Supreme Court of the United StatesMARIE PFAU, PETITIONERv.WILLIAM REED, DIRECTOR,DEFENSE CONTRACT AUDIT AGENCYON PETITION FOR A WRIT OF CERTIORARITO THE UNITED STATES COURT OF APPEALSFOR THE FIFTH CIRCUITBRIEF FOR THE RESPONDENT IN OPPOSITIONSETH P. WAXMANSolicitor GeneralCounsel of RecordDAVID W. OGDENActing Assistant Attorney GeneralMARLEIGH D. DOVERWENDY M. KEATSAttorneysDepartment of JusticeWashington, D.C. 20530-0001(202) 514-2217QUESTION PRESENTEDWhether Title VII of the Civil Rights Act of 1964, 42 U.S.C. 2000e et seq., preempts petitioner's claims under the Federal Tort Claims Act and Texas state law for intentional infliction of emotional distress.In the Supreme Court of the United StatesNo. 98-1846MARIE PFAU, PETITIONERv.WILLIAM REED, DIRECTOR,DEFENSE CONTRACT AUDIT AGENCYON PETITION FOR A WRIT OF CERTIORARITO THE UNITED STATES COURT OF APPEALSFOR THE FIFTH CIRCUITBRIEF FOR THE RESPONDENT IN OPPOSITIONOPINIONS BELOWThe opinion of the court of appeals (Pet. App. 38a-39a) is reported at 167 F. 3d 228. The initial opinion of the court of appeals (Pet. App. 9a-33a) is reported at 125 F.3d 927.JURISDICTIONThe judgment of the court of appeals was entered on February 8, 1999. The petition for a writ of certiorari was filed on May 10, 1999 (a Monday). The jurisdiction of this Court is invoked under 28 U.S.C. 1254(1).STATEMENT1. Petitioner is a former employee of the Defense Contract Audit Agency (DCAA). Pet. App. 10a. Petitioner worked on an audit team where Pete Gonzales was her first-line supervisor. Ibid. Petitioner alleges that, when she first became a member of that team, Gonzales made lewd and suggestive comments to her and requested sexually provocative behavior from her. Ibid. Petitioner further alleges that Gonzales requested that she take him on a trip with her, made sexual advances she rejected, asked to go on vacations with her at her expense, and asked her for money on several occasions. Ibid. Petitioner also alleges that Gonzales called her, appeared at her home, and insisted that they become sexually involved. Id. at 11a.Petitioner alleges that, after she complained to management that Gonzales had sexually harassed her, Gonzales retaliated against her. Pet. App. 11a. Among other things, she alleges that Gonzales gave her inappropriate work assignments, denied her training, and denied her request for sick leave. Ibid. Petitioner was ultimately fired from her job. Id. at 10a.2. Petitioner filed suit in the United States District Court for the Western District of Texas against the Director of the DCAA and Gonzales among others. Pet. App. 12a. Petitioner alleged that Gonzales had subjected her to sexual harassment for which the DCAA was liable under Title VII of the Civil Rights Act of 1964, 42 U.S.C. 2000e et seq., and the Civil Rights Act of 1991, Pub. L. No. 102-166, § 102, 105 Stat. 1071 (42 U.S.C. 1981a). Pet. App. 12a. She also alleged against Gonzales a state law tort claim of intentional infliction of emotional distress. Ibid.The district court dismissed petitioner's claim of intentional infliction of emotional distress. Pet. App. 4a-8a. The court held that Title VII and the Civil Service Reform Act of 1978 (CSRA), Pub. L. No. 95-454, 92 Stat. 1111, preempted that claim. Pet. App. 5a-7a. Petitioner amended her complaint to add the United States as a defendant and to assert a claim of intentional infliction of emotional distress under the Federal Tort Claims Act (FTCA). Id. at 12a. Petitioner also reasserted her state law tort claim against Gonzales. Ibid. The district court dismissed both tort claims, once again relying on the preemptive force of Title VII and the CSRA. Ibid. The court then granted summary judgment in favor of the government on petitioner's Title VII claim, id. at 13a, holding that Gonzales did not qualify as petitioner's "employer" for purposes of imputing liability to the DCAA under Title VII, and that petitioner's evidence failed to raise an inference that DCAA had actual or constructive knowledge of Gonzales's alleged harassment prior to petitioner's formal complaint or that it had failed to take appropriate action following the complaint. See id. at 20a.3. The court of appeals affirmed. Pet. App. 9a-33a. The court held that, under circuit precedent, "[w]hen the same set of facts supports a Title VII claim and a non-Title VII claim against a federal employer, Title VII preempts the non-Title VII claim." Id. at 15a. Finding that the same set of facts supported petitioner's Title VII claim and her intentional infliction of emotional distress claims, the court concluded that Title VII preempted petitioner's intentional infliction of emotional distress claims. Id. at 15a-18a. The court rejected petitioner's contention that her tort claims are distinct from her Title VII claim, because some of the alleged conduct occurred away from the office and after business hours. Id. at 16a. The court explained that, under Meritor Savings Bank, FSB v. Vinson, 477 U.S. 57 (1986), those factual allegations support her Title VII claim. Pet. App. 16a.The court of appeals also upheld the district court's grant of summary judgment dismissing petitioner's Title VII claim. Pet. App. 18a-33a. The court held that Gonzales was not petitioner's "employer" for purposes of Title VII under existing precedent, id. at 21a-26a, and that petitioner's evidence raised no genuine issue of material fact with regard to DCAA's knowledge of the alleged harassment prior to petitioner's formal complaint or the adequacy of its response following the complaint, id. at 26a-33a.4. Petitioner sought certiorari, and this Court granted the petition, vacated the court of appeals' judgment, and remanded the case for further consideration in light of the intervening decisions in Faragher v. City of Boca Raton, 118 S. Ct. 2275 (1998), and Burlington Indus., Inc. v. Ellerth, 118 S. Ct. 2257 (1998). 119 S. Ct. 32 (1998). Those cases established the standards governing employer liability under Title VII for harassment committed by a supervisor.On remand, the court of appeals reinstated the part of its prior opinion concerning Title VII's preemption of petitioner's claims of intentional infliction of emotional distress and reaffirmed the dismissal of those claims. Pet. App. 38a-39a. The court remanded petitioner's Title VII sexual harassment claim for further consideration and fact-finding by the district court, in light of Faragher and Ellerth. Id. at 39a.ARGUMENT1. Petitioner seeks review of the court of appeals' holding that Title VII preempts her claims under the Federal Torts Claims Act (FTCA) and Texas state law for intentional infliction of emotional distress. Because the court of appeals remanded the case for consideration of petitioner's Title VII claim, however, the case is in an interlocutory posture. This Court ordinarily refuses to grant review when the decision being challenged is interlocutory. See Brotherhood of Locomotive Firemen v. Bangor & Aroostook R.R., 389 U.S. 327, 328 (1967) ("because the Court of Appeals remanded the case, it is not yet ripe for review by this Court"); see also Virginia Military Inst. v. United States, 508 U.S. 946 (1993) (Scalia, J., respecting the denial of certiorari) (this Court "generally await[s] final judgment in the lower courts before exercising [its] certiorari jurisdiction").There is no reason to depart from the Court's usual practice here. Petitioner's challenge to the court of appeals' preemption ruling depends on her assertion that her claims involving intentional infliction of emotional distress are distinct from her Title VII claim. The court of appeals' remand order contemplates additional discovery and fact-finding on her Title VII claim that could cast further light on its relationship to her claims of intentional infliction of emotional distress. Such facts could provide additional support for the court of appeals' conclusion that all of the alleged conduct falls within petitioner's Title VII claim, or could cause the court to revisit that conclusion. If a live dispute persists on that issue after remand, petitioner will retain her right to present to this Court any claim she presents here so long as she has preserved it. See, e.g., Urie v. Thompson, 337 U.S. 163, 172-173 (1949); Robert L. Stern et al., Supreme Court Practice § 4.18, at 198 (7th ed. 1993).2. Even if petitioner's challenge to the court of appeals' preemption ruling were ripe, it would not warrant review. In Brown v. GSA, 425 U.S. 820, 835 (1976), this Court held that Title VII "provides the exclusive judicial remedy for claims of discrimination in federal employment." The court of appeals in this case interpreted that holding to mean that "[w]hen the same set of facts supports a Title VII claim and a non-Title VII claim against a federal employer, Title VII preempts the non-Title VII claim." Pet. App. 15a. Finding that the same set of facts supported petitioner's Title VII claim and her intentional infliction claims, the court concluded that Title VII preempted petitioner's intentional infliction claims. Id. at 14a-18a.a. Petitioner contends (Pet. 9-11) that some of the factual allegations upon which she relies to support her intentional infliction claims do not support her Title VII claim. That fact-bound challenge to the decision below does not raise any issue of general importance and therefore does not warrant review.In any event, the court of appeals correctly concluded that all of petitioner's allegations would be relevant in establishing a Title VII claim. Petitioner contends (Pet. 9) that some of the factual allegations upon which she relied to support her intentional infliction claims are not relevant to her Title VII claim, because they do not involve "sexual" conduct. Those allegations are that Gonzales requested money from petitioner, sought joint vacations with her, and made phone calls to her house. Title VII, however, does not require proof that harassing conduct is of a sexual nature. Rather, "any harassment or other unequal treatment of an employee or group of employees that would not occur but for the sex of the employee or employees may, if sufficiently severe or pervasive, comprise an illegal condition of employment under Title VII." McKinney v. Dole, 765 F.2d 1129, 1138 (D.C. Cir. 1985); see also Hall v. Gus Constr. Co., 842 F.2d 1010, 1014 (8th Cir. 1988) ("[i]ntimidation and hostility toward women because they are women can obviously result from conduct other than explicit sexual advances"); Hicks v. Gates Rubber Co., 833 F.2d 1406, 1415 (10th Cir. 1987) ("evidence of threats of physical violence and incidents of verbal abuse" would be considered along with evidence of sexual harassment in determining hostile work environment claim).Applying that standard, Gonzales's alleged money requests, vacation demands, and phone calls are relevant to petitioner's Title VII claim. Those incidents allegedly occurred during the same time period that Gonzales engaged in repeated sexual advances, made lewd and suggestive comments, and demanded sexual relations. Pet. App. 10a-11a. Whether or not the incidents were non-sexual, they could help to establish that petitioner was subjected to a pattern of harassment that would not have occurred but for her sex and that was sufficiently severe and pervasive to affect the terms and conditions of her employment. The court of appeals therefore correctly concluded that those incidents are relevant to petitioner's Title VII claim.Petitioner also contends (Pet. 10) that some of the conduct upon which she relies for her intentional infliction claims is not relevant to her Title VII claim, because it occurred after work hours, outside of work premises. When a supervisor harasses a subordinate after work hours and outside work premises, however, it can have a substantial effect on the way that employee experiences her working environment. Such evidence is therefore relevant in establishing a Title VII hostile work environment claim. See Meritor Sav. Bank, FSB v. Vinson, 477 U.S. 57, 60 (1986) (plaintiff's allegation that her supervisor invited her to dinner, suggested at dinner that they go to a motel for sexual relations, and made repeated demands for sexual favors both during and after business hours, formed part of the basis for a Title VII sexual harassment claim).In sum, all of the evidence upon which petitioner relies for her intentional infliction claims is also relevant to her Title VII claim. Petitioner's contention to the contrary is without merit.b. Petitioner also contends (Pet. 7, 12-15) that the court of appeals' "same facts" test is not the correct standard for judging the preemptive force of Title VII, and that it conflicts with Ninth Circuit decisions holding that Title VII does not preempt FTCA and state law causes of action that involve a "highly personal violation beyond the meaning of discrimination." Brock v. United States, 64 F.3d 1421, 1424 (1995); see also Arnold v. United States, 816 F.2d 1306, 1311-1312 (1987); Otto v. Heckler, 781 F.2d 754, 756-758 (1986). For several reasons, that contention does not warrant review in this case.First, only the Fifth and Ninth circuits have addressed the extent to which Title VII preempts FTCA and state law tort claims. That issue would benefit from further ventilation in the regional courts of appeals.Second, the Ninth Circuit's decisions have all involved particularly aggravated forms of conduct; in no case has the sole claim been one of intentional infliction of emotional distress. Brock, 64 F.3d at 1421 (rape and assault); Arnold, 816 F.2d at 1312 (assault, battery, and false imprisonment); Otto, 781 F.2d at 755, 757-758 (stalking and placing in fear of sexual assault resulting in a miscarriage). It is unclear whether the Ninth Circuit would conclude that intentional infliction of emotional distress without more is the kind of "highly personal violation beyond the meaning of discrimination" that is not preempted by Title VII.Third, all of the Ninth Circuit cases involved claims based on conduct that predated the 1991 amendment to Title VII, which provides that victims of intentional discrimination may seek compensatory relief. The 1991 amendment was intended to afford the victims of sexual harassment compensation for injuries to "their mental, physical, and emotional health, to their self-respect and dignity, and for other consequential harms." 137 Cong. Rec. 30,661 (1991) (section-by-section analysis of Rep. Edwards). As a result of that amendment, Title VII now provides compensation for the same kinds of injuries that are alleged as a basis for claims of intentional infliction of emotional distress. The Ninth Circuit has previously held that Title VII preempted a state law tort suit for defamation when the plaintiff sought compensation for the same "injuries cognizable and remediable under Title VII." Otto, 781 F.2d at 757. In light of the 1991 amendment, the Ninth Circuit may reach a similar conclusion with respect to Title VII's effect on the tort of intentional infliction of emotional distress.Fourth, in deciding the preemption question, the Ninth Circuit did not have the benefit of this Court's decisions in Faragher and Ellerth establishing the principles that govern the extent to which an employer is liable under Title VII for harassment committed by a supervisor. Because those decisions help to define the reach of Title VII, they may also affect the scope of Title VII's preemptive effect.Finally, employer liability decisions in the wake of Faragher and Ellerth may reduce the practical importance of the preemption question, particularly in conjunction with the 1991 amendment. Since plaintiff employees can more readily establish employer liability for supervisory harassment and can now obtain compensatory relief for a violation, they have less incentive than before to pursue FTCA and state law tort claims. Experience under the new rules for determining employer liability for supervisory harassment is necessary before an assessment can be made concerning the continuing importance of the preemption issue.For all these reasons, the question concerning the correct legal standard for determining Title VII's preemptive effect on FTCA and state tort law therefore does not warrant review in this case.CONCLUSIONThe petition for a writ of certiorari should be denied.Respectfully submitted.SETH P. WAXMANSolicitor GeneralDAVID W. OGDENActing Assistant Attorney GeneralMARLEIGH D. DOVERWENDY M. KEATSAttorneysAUGUST 1999 Type: Petition Stage Response Briefs: 98-1846.resp.pdf	https://www.justice.gov/osg/brief/pfau-v-reed-opposition-0
ROAD MAP MORIEZ : maps of Moriez 04170 ROAD MAP MORIEZ : road map, relief map, administrative maps, geographic information and location of Moriez on the map of France 04170 Alpes-de-Haute-Provence. Road map Moriez http://www.cartesfrance.fr Maps of Moriez Moriez Hotel Road map Map Photos Weather Population Housing <table><tbody><tr><td> Here are several maps of Moriez. You will find theroad map of Moriezwith a routing module. This dynamic road map of France (in sexagesimal coordinates WGS84) is centered on the town of Moriez. The location of Moriez in France is shown below on several maps of France. The mapping system of thesebase maps of Moriezis the french mapping system: Lamber93.These base maps of Moriez are reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code. You will find thehotels near Moriezon this map. Book your hotel near Moriez today, Monday 17 July for the best price, reservation and cancellation free of chargethanks to our partner Booking.com, leader in on-line hotels reservation. To help you choose your destination here is:- pictures of Moriez :photo Moriez- the map of Moriez :map Moriez- the hotels map of Moriez : Moriez hotels map </td><td><table><tbody><tr><td><table><tbody><tr><td colspan=3> I like Moriez !</td></tr><tr><td></td><td></td><td></td></tr></tbody></table></td></tr><tr><td> </td></tr></tbody></table></td></tr></tbody></table> Road map of Moriez <table><tbody><tr><td> Get directionsto Moriez with ourrouting module. You will find thehotels near Moriezwith the search box at right. Book your hotel near Moriez today, Monday 17 July for the best price, reservation and cancellation free of charge thanks to our partner Booking.com, leader in on-line hotels reservation. To help you choose your destination here is: - pictures of Moriez : photo Moriez - the map of Moriez : map Moriez - the hotels map of Moriez : Moriez hotels map - the location of Moriez in France is shown below on several maps of France. The mapping system of thesebase maps of Moriezis the french mapping system: Lamber93. These base maps of Moriez are reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code.</td><td> Search Hotels Book now, Best Price Guaranteed ! Where ? Moriez Check-in date 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June 2023 July 2023 August 2023 September 2023 October 2023 November 2023 December 2023 January 2024 February 2024 March 2024 April 2024 May 2024<here is a image 3e7ad46591489709-a44d4ff1ef498f1b> Check-out date 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June 2023 July 2023 August 2023 September 2023 October 2023 November 2023 December 2023 January 2024 February 2024 March 2024 April 2024 May 2024<here is a image 3e7ad46591489709-a44d4ff1ef498f1b> on I have no specific dates Search</td></tr></tbody></table> Relief map of Moriez <table><tbody><tr><td colspan=2> Moriez on the relief France map in Lambert 93 coordinates </td></tr><tr><td> <here is a image c67fbca86ad520b3-7a9b4ed288b62a2b> See the map of Moriez in full screen (1000 x 949)</td><td> At left, here is the location of Moriez on the relief France map in Lambert 93 coordinates. Here are the altitudes of Moriez :- Town hall altitude of Moriez is 920 meters- Minimum altitude of Moriez is 820 meters- Maximum altitude of Moriez is 1 700 meters- Medium altitude of Moriez is 1 260 meters Here are the altitudes ofDigne-les-Bains, prefecture ofAlpes-de-Haute-Provencedepartment:- Town hall altitude of Digne-les-Bains is 600 meters- Minimum altitude of Digne-les-Bains is 524 meters- Maximum altitude of Digne-les-Bains is 1 731 meters- Medium altitude of Digne-les-Bains is 1 128 meters Here are the altitudes of the french biggest cities: Paris: 33 meters Marseille: 20 meters Lyon: 237 meters Toulouse: 146 meters Nice: 10 meters Nantes: 20 meters Strasbourg: 144 meters Montpellier: 35 meters Bordeaux: 16 meters Lille: 20 meters Rennes: 35 meters Reims: 83 meters Click here for the terrain map of Moriez in full screen: Relief map of Moriez</td></tr><tr><td colspan=2> This relief map of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Relief map of Moriez" src="https://www.map-france.com/town-map/04/04133/france-map-relief-big-cities-Moriez.jpg" /></td></tr><tr><td colspan=2> Terrain base map of Moriez </td></tr><tr><td> <here is a image 3f0daa4dfae1d709-6abfbf94d388b46b> See the terrain base map of Moriez in full screen (1000 x 949)</td><td> Small relief base map of Moriez <here is a image aacb95d8d426ef36-4963f116b00cad42> </td></tr><tr><td colspan=2> This base map of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Base relief map of Moriez" src="https://www.map-france.com/town-map/04/04133/france-map-relief-Moriez.jpg" /></td></tr></tbody></table> Administrative maps of Moriez <table><tbody><tr><td colspan=2> Map of Moriezwith regions and prefectures </td></tr><tr><td> <here is a image d58bee72257c591d-957b4b03e0690aed> View the map of Moriez in full screen (1000 x 949)</td><td> You will find the location of Moriez on the map of France of regions in Lambert 93 coordinates. The town of Moriezis located in the department ofAlpes-de-Haute-Provenceof the french regionProvence-Alpes-Côte d'Azur. Geographical sexagesimal coordinates / GPS(WGS84):Latitude: 43° 57' 42'' NorthLongitude: 06° 28' 15'' East Geographical decimal coordinates:Latitude: 43.963 degrees (43.963° North)Longitude: 6.472 degrees (6.472° East) Lambert 93 coordinates:X: 9 785 hectometersY: 63 242 hectometers Lambert 2 coordinates:X: 9 320 hectometersY: 18 932 hectometers Below, the geographical coordinates ofDigne-les-Bains, prefecture of theAlpes-de-Haute-Provencedepartment: Geographical sexagesimal coordinates / GPS(WGS84):Latitude: 44° 05' 33'' NorthLongitude: 06° 14' 08'' East Geographical decimal coordinates:Latitude: 44.095 degrees (44.095° North)Longitude: 6.235 degrees (6.235° East) Lambert 93 coordinates:X: 9 590 hectometersY: 63 379 hectometers Lambert 2 coordinates:X: 9 124 hectometersY: 19 068 hectometers</td></tr><tr><td colspan=2> This map of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Moriez on the map of France with regions" src="https://www.map-france.com/town-map/04/04133/administrative-france-map-regions-Moriez.jpg" /></td></tr><tr><td colspan=2> Map of Moriezwith departments and prefectures </td></tr><tr><td> <here is a image fcc568d39319d14e-7b25aa59edebf019> Voir la carte of Moriez en grand format (1000 x 949)</td><td> Here is the localization of Moriez on the France map of departments in Lambert 93 coordinates. The city of Moriez is shown on the map by a red point. The town of Moriezis located in the department ofAlpes-de-Haute-Provenceof the french regionProvence-Alpes-Côte d'Azur. The latitude of Moriez is 43.963 degrees North.The longitude of Moriez is 6.472 degrees East. Here the distance between Moriez and the biggest cities of France: Distances are calculated as the crow flies (orthodromic distance) Distance between Moriez andParis: 629.10 kilometersDistance between Moriez andMarseille: 113.57 kilometersDistance between Moriez andLyon: 237.64 kilometersDistance between Moriez andToulouse: 404.75 kilometersDistance between Moriez andNice: 69.48 kilometersDistance between Moriez andNantes: 720.77 kilometersDistance between Moriez andStrasbourg: 522.86 kilometersDistance between Moriez andMontpellier: 212.01 kilometersDistance between Moriez andBordeaux: 567.65 kilometersDistance between Moriez andLille: 784.07 kilometersDistance between Moriez andRennes: 778.71 kilometersDistance between Moriez andReims: 617.03 kilometers Clic here to see the map of Moriez in full screen: Moriez on the map of french departments</td></tr><tr><td colspan=2> This administrative map of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Moriez on the map of France with departments" src="https://www.map-france.com/town-map/04/04133/administrative-france-map-departements-Moriez.jpg" /></td></tr><tr><td colspan=2> Administrative base maps of Moriez </td></tr><tr><td> <here is a image 3c5eb0c07fd34953-cb8a4e52f3d97e6c> See the administrative base map of Moriez in full screen (1000 x 949)</td><td> Small administrative base map of Moriez <here is a image 1b1b256fbe423eec-08044a766c3d6443> </td></tr><tr><td colspan=2> This base map administrative of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Base administrative map of Moriez" src="https://www.map-france.com/town-map/04/04133/administrative-france-map-Moriez.jpg" /></td></tr></tbody></table> Moriez on the municipalities map of France <table><tbody><tr><td colspan=2> Each black point on the following maps is a municipality of France. These maps are made from all points of all municipalities of France.The red point show the location of the town hall of the Moriez municipality.</td></tr><tr><td> Moriezon the municipalities map of France <here is a image dbec019c7215b225-5ae59c2d3ccc6e11> See the map of Moriez in full screen (1000 x 949)</td><td> Moriezon the municipalities map of France <here is a image bea4ffebcf4e8b82-4a6fc982e96ac77c> See the map of Moriez in full screen (1000 x 949)</td></tr><tr><td colspan=2> This base map of Moriez is reusable and modifiable by making a link to this page of the Map-France.com website or by using the given code : <img alt="Moriez on the municipalities map of France" src="https://www.map-france.com/town-map/04/04133/france-map-town-Moriez.jpg" /></td></tr></tbody></table>	https://www.map-france.com/Moriez-04170/road-map-Moriez.html
HUBBUK 2016, Baskerville Hall, June 16-19 - Page 5 - Horizons Unlimited - The HUBB I am pleased to say that our club http://theoverlander.org Are attending in their 12 ton Overland trucks. Should be a great weekend. Neil Cloud 9 is Born Adventure is what you make itby Simon McCarthy."Alternative" bridge near Salavan, Laos # 61 13 May 2016 Neil H Registered Users HUBB regular Join Date: May 2007 Location: Essex Uk Posts: 72 I am pleased to say that our club http://theoverlander.org Are attending in their 12 ton Overland trucks. Should be a great weekend. Neil Cloud 9 is Born Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 62 13 May 2016 backofbeyond Registered Users Veteran HUBBer Join Date: Apr 2005 Location: Oxford UK Posts: 2,066 Quote: Originally Posted by Grant Johnson We don't have hundreds of vendors and a wall of death for entertainment, No wall of death! That's a disappointment, I was quite looking forward to having a go at that. Never mind, I can console myself with a burger or something. There is a McDonalds, right? (Actually, given how useful McDonalds wi-fi is when you're on the road maybe they ought to be there.) Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 63 17 May 2016 Jo_carter Registered Users HUBB regular Join Date: May 2012 Location: Manchester Posts: 23 Hubb UK facebook page have you seen it? Hi All, We are posting lots of new information (presenters, sponsors, bands....and lots more) on the UK Hubb facebook page if your on facebook have a look & you are to share with your friends & clubs https://www.facebook.com/events/2029...tive_tab=posts All the best Jo Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 64 21 May 2016 Swampy46 Registered Users New on the HUBB Join Date: Jul 2011 Location: South Devon Posts: 5 Confirmation Hi all. Stupid question, I registered and paid for the UK event in January, was there a confirmation e mail, the reason I ask is that some of my folders in my e mail have disappeared (thanks Virgin). Is there a way of checking I am on the list? I am just trying to be organised for a change. Also, my son (17) has seen the site and wants to come, I am assuming I need to register him separately. Any help for this newbie would be appreciated. Thanks. Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 65 21 May 2016 Grant Johnson HU Founder Veteran HUBBer Join Date: Dec 1997 Location: BC Canada Posts: 7,020 Quote: Originally Posted by Swampy46 Hi all. Stupid question, I registered and paid for the UK event in January, was there a confirmation e mail, the reason I ask is that some of my folders in my e mail have disappeared (thanks Virgin). Is there a way of checking I am on the list? I am just trying to be organised for a change. Also, my son (17) has seen the site and wants to come, I am assuming I need to register him separately. Any help for this newbie would be appreciated. Thanks. Yes, you should get a confirmation email within seconds of submitting. If not, the email address you entered is bad, or Virgin lost it! (there's something funny there, just not sure what...) Yes, you ARE registered and even paid! Yes, EVERYONE registers separately, it's the only way we can be sure to get it right. How's that - three yeses! oops better make that three see you there Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ __________________ Grant Johnson Seek , and ye shall find . ------------------------ Inspiring, Informing and Connecting travellers since 1997! www.HorizonsUnlimited.com # 66 21 May 2016 Swampy46 Registered Users New on the HUBB Join Date: Jul 2011 Location: South Devon Posts: 5 Thanks Grant, I'll get my lad registered, I am assuming my entry will be ok without a copy of the e mail. Really looking forward to it. Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 67 22 May 2016 Grant Johnson HU Founder Veteran HUBBer Join Date: Dec 1997 Location: BC Canada Posts: 7,020 Quote: Originally Posted by Swampy46 Thanks Grant, I'll get my lad registered, I am assuming my entry will be ok without a copy of the e mail. Really looking forward to it. Paper not required - save the planet! Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ __________________ Grant Johnson Seek , and ye shall find . ------------------------ Inspiring, Informing and Connecting travellers since 1997! www.HorizonsUnlimited.com # 68 22 May 2016 OTRA Registered Users HUBB regular Join Date: Aug 2013 Location: DIEPPE - NORMANDY - FRANCE Posts: 77 Hi Gipsy Quote: Originally Posted by The_gypsy Whimp, Ya havn't lived untillya slid down a dirt track sideways on a road bike. Why does everyone think that a road bike is for roads and only an adventure bike is for adventures. Sean did the Pan American on a 100cc Honda Cub. There are plenty of instances of like minded souls doing the same. I have decided to do a "JoinTheDots" tour to all the places I've missed out on over the years. But not decided which bike to use. So will be discussing this over a glass of something at the HU. Hope Ted is there to give us some advice. Look forward to meeting ya'all. I'll be there this year, arriving thursday afternoon with sheese and wine (perfect contact kit ) hope to see you there. Are you coming to the french one in september? It's gonna be a fantastic meeting,as usual (well done Pongo! ) see you all very soon Ride safe and cool Roger Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ Sloth505 likes this. # 69 22 May 2016 Swampy46 Registered Users New on the HUBB Join Date: Jul 2011 Location: South Devon Posts: 5 Son is registered and paid for, just a thought, my tent was going on my back seat so where shall I put him? Neil, had a look at your site, trucks look awesome. Can't wait to get some ideas for my next trip. Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 71 22 May 2016 deenewcastle Registered Users HUBB regular Join Date: Jun 2010 Location: Newcastle upon Tyne Posts: 66 My party of 3 have had to change our plans quite a bit. We can no longer come using the bikes we planned, and will be one car and one 4x4. We will also be arriving very late Thursday night rather than Thursday afternoon. What time will Reception close on Thursday and re-open Friday morning? Deana (getting really really excited to be at HU again) Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 72 23 May 2016 Glynn Roberts Lifetime Member Veteran HUBBer Join Date: Oct 2000 Location: Nottingham England Posts: 144 Reception will probably close around 10pm every evening. Glynn Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ deenewcastle likes this. # 73 23 May 2016 Glynn Roberts Lifetime Member Veteran HUBBer Join Date: Oct 2000 Location: Nottingham England Posts: 144 The HUBB has always been traditionally held the same weekend. Its sometimes changed due the venues commitments but middle of June is the ideal date! Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 74 24 May 2016 rms Registered Users New on the HUBB Join Date: Oct 2012 Location: Near Wakefield, W Yorks, UK Posts: 11 Quote: Originally Posted by LiamMcILhone Now that you are all booked up and planning to attend HUBBUK (you haven't yet?........then why not ) why not consider extending your hard-earned break and coming over to the HU Ireland event taking place the following weekend in Enniskillen (24/26 June ) Details for the weekend can be found at: http://www.horizonsunlimited.com/events/ireland-2016 If you enjoy the HUBBUK meeting (and you will !) then you will certainly enjoy the Ireland meeting with the added bonus of having the opportunity to explore some of the great scenery and roads in Ireland. And need I mention the Irish hospitality, the "craic" and just perhaps, Guinness Whichever HU event you choose to attend, where ever it may be in the world, you can be assured of a great weekend in the company of like-minded people. Slainte from Ireland Hi Liam, We'd have loved to pop over to do some more of the Wild Atlantic Way in our Land Rover Carawagons and join you at HU Ireland again, but too many things have conspired to get in our way this year as I'm sure Steve will have told you A shame as we loved the informal, more 'intimate' meeting there last year, but don't worry - we're planning on trying again for next year Hopefully see you then Cheers, Robin Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ # 75 24 May 2016 M.Aziz Registered Users New on the HUBB Join Date: Apr 2014 Location: Sydney, Australia Posts: 14 Hello all, i don't know if this is the place to put this but here goes. My bike is currently on a ship inbound from Sydney to the UK, i have been informed it won't be here until the 23rd of June. All my camping gear is in the crate with the bike, i have registered and paid for the HUBB UK event but with no easy way to get there or a tent, its minimum 11 hours to get there from London via public transport and walking if i don't get lost. Is anyone from London heading over and back via their car and would allow me to tag along for fuel money? Also I'm looking to rent at least a tent if anyone won't be using their one for the event. I can find cheap used tents on eBay, getting to the event is more what I need help with. Cheers and looking forward to meeting some of you at the event Share Share this post on Digg Del.icio.us Twitter Share on Facebook Reddit! Share on Google+ Sloth505 likes this.	https://www.horizonsunlimited.com/hubb/hu-travellers-meetings-uk/hubbuk-2016-baskerville-hall-june-85248-5
Exciting spiral arms in protoplanetary discs from flybys \| Request PDF Request PDF \| Exciting spiral arms in protoplanetary discs from flybys \| Spiral arms are observed in numerous protoplanetary discs. These spiral arms can be excited by companions, either on bound or unbound orbits. We... \| Find, read and cite all the research you need on ResearchGate Exciting spiral arms in protoplanetary discs from flybys March 2023 Monthly Notices of the Royal Astronomical Society521(3) DOI: 10.1093/mnras/stad742 Authors: <here is a image f2f62089ad6ff61c-d19572ff1996375a> Jeremy L Smallwood Jeremy L Smallwood <here is a image f2f62089ad6ff61c-d19572ff1996375a> Chao-Chin Yang Chao-Chin Yang <here is a image f2f62089ad6ff61c-d19572ff1996375a> Zhaohuan Zhu Zhaohuan Zhu Rebecca G Martin Rebecca G Martin Abstract Spiral arms are observed in numerous protoplanetary discs. These spiral arms can be excited by companions, either on bound or unbound orbits. We simulate a scenario where an unbound perturber, i.e. a flyby, excites spiral arms during a periastron passage. We run three-dimensional hydrodynamical simulations of a parabolic flyby encountering a gaseous protoplanetary disc. The perturber mass ranges from $10\, \rm M_J$ to $1\, \rm {\rm M}_{\odot }$. The perturber excites a two-armed spiral structure, with a more prominent spiral feature for higher mass perturbers. The two arms evolve over time, eventually winding up, consistent with previous works. We focus on analysing the pattern speed and pitch angle of these spirals during the whole process. The initial pattern speed of the two arms are close to the angular velocity of the perturber at periastron, and then it decreases over time. The pitch angle also decreases over time as the spiral winds up. The spirals disappear after several local orbital times. An inclined prograde orbit flyby induces similar disc substructures as a coplanar flyby. A solar-mass flyby event causes increased eccentricity growth in the protoplanetary disc, leading to an eccentric disc structure which dampens over time. The spirals’ morphology and the disc eccentricity can be used to search for potential unbound stars or planets around discs where a flyby is suspected. Future disc observations at high resolution and dedicated surveys will help to constrain the frequency of such stellar encounters in nearby star-forming regions. <here is a image 175d1bd4b7439d95-754927124da715c1> Stellar Flyby Analysis for Spiral Arm Hosts with Gaia DR3 Article Full-text available Dec 2022 Linling Shuai Bin Ren Dimitri Mawet <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Ruobing Dong Scattered-light imaging studies have detected nearly two dozen spiral arm systems in circumstellar disks, yet the formation mechanisms for most of them are still under debate. Although existing studies can use motion measurements to distinguish leading mechanisms such as planet–disk interaction and disk self-gravity, close-in stellar flybys can induce short-lived spirals and even excite arm-driving planets into highly eccentric orbits. With unprecedented stellar location and proper-motion measurements from Gaia Data Release 3 (DR3), here we study for known spiral arm systems their flyby history with their stellar neighbors by formulating an analytical on-sky flyby framework. For stellar neighbors currently located within 10 pc of the spiral hosts, we restrict the flyby time to within the past 10 ⁴ yr and the flyby distance to within 10 times the disk extent in scattered light. Among a total of 12,570 neighbors that are identified in Gaia DR3 for 20 spiral systems, we do not identify credible flyby candidates for isolated systems. Our analysis suggests that a close-in recent flyby is not the dominant formation mechanism for isolated spiral systems in scattered light. The SPHERE view of three interacting twin disc systems in polarised light Article Full-text available Nov 2022 MON NOT R ASTRON SOC Philipp Weber G. Guidi Julien H. Girard <here is a image 01d52cef581ccf05-adba71de9fce25d8> Sebastian Perez Dense stellar environments as hosts of ongoing star formation increase the probability of gravitational encounters among stellar systems during the early stages of evolution. Stellar interaction may occur through non-recurring, hyperbolic or parabolic passages (a so-called ‘fly-by’), through secular binary evolution, or through binary capture. In all three scenarios, the strong gravitational perturbation is expected to manifest itself in the disc structures around the individual stars. Here, we present near-infrared polarised light observations that were taken with the SPHERE/IRDIS instrument of three known interacting twin-disc systems: AS 205, EM* SR 24, and FU Orionis. The scattered light exposes spirals likely caused by the gravitational interaction. On a larger scale, we observe connecting filaments between the stars. We analyse their very complex polarised intensity and put particular attention to the presence of multiple light sources in these systems. The local angle of linear polarisation indicates the source whose light dominates the scattering process from the bridging region between the two stars. Further, we show that the polarised intensity from scattering with multiple relevant light sources results from an incoherent summation of the individuals’ contribution. This can produce nulls of polarised intensity in an image, as potentially observed in AS 205. We discuss the geometry and content of the systems by comparing the polarised light observations with other data at similar resolution, namely with ALMA continuum and gas emission. Collective observational data can constrain the systems’ geometry and stellar trajectories, with the important potential to differentiate between dynamical scenarios of stellar interaction. Observing Planet-driven Dust Spirals with ALMA Article Full-text available May 2022 <here is a image 715bb4d0340b1f58-bd123d0888550ae2> Jessica Speedie Richard A. Booth Ruobing Dong Atacama Large Millimeter/submillimeter Array (ALMA) continuum observations of thermal emission from the dust component of protoplanetary disks have revealed an abundance of substructures that may be interpreted as evidence for embedded planets, but planet-driven spiral arms—perhaps one of the most compelling lines of evidence—have proven comparatively elusive. In this work, we test the capabilities of ALMA to detect the planet-driven spiral signal in continuum emission. Carrying out hydrodynamic simulations and radiative transfer calculations, we present synthetic Band 7 continuum images for a wide range of disk and observing conditions. We show that thermal mass planets at tens of astronomical units typically drive spirals detectable within a few hours of integration time, and the detectable planet mass may be as low as ∼Neptune mass (0.3 M th ). The grains probed by ALMA form spirals morphologically identical to the underlying gas spiral. The temperature of the dust spiral is crucial in determining its contrast, and spirals are easier to detect in disks with an adiabatic equation of state and longer cooling times. Resolving the spiral is not necessary for its detection; with the help of residual maps, the optimal beam size is a few times the spiral width at a constant noise level. Finally, we show how the presence of gaps and rings can impair our ability to recognize colocated spirals. Our work demonstrates the planet-finding potential of the current design specification of ALMA, and suggests that observing capability is not the bottleneck in searching for spirals induced by thermal mass planets. A Highly Settled Disk around Oph163131 Article Full-text available May 2022 M. Villenave Karl R. Stapelfeldt <here is a image c6f7f41bbe259cd0-6c1e88b84b343c4c> G. Duchene C. Pinte High dust density in the midplane of protoplanetary disks is favorable for efficient grain growth and can allow fast formation of planetesimals and planets, before disks dissipate. Vertical settling and dust trapping in pressure maxima are two mechanisms allowing dust to concentrate in geometrically thin and high-density regions. In this work, we aim to study these mechanisms in the highly inclined protoplanetary disk SSTC2D J163131.2-242627 (Oph 163131, i ∼ 84°). We present new high-angular-resolution continuum and ¹² CO ALMA observations of Oph 163131. The gas emission appears significantly more extended in the vertical and radial direction compared to the dust emission, consistent with vertical settling and possibly radial drift. In addition, the new continuum observations reveal two clear rings. The outer ring, located at ∼100 au, is well-resolved in the observations, allowing us to put stringent constraints on the vertical extent of millimeter dust particles. We model the disk using radiative transfer and find that the scale height of millimeter-sized grains is 0.5 au or less at 100 au from the central star. This value is about one order of magnitude smaller than the scale height of smaller micron-sized dust grains constrained by previous modeling, which implies that efficient settling of the large grains is occurring in the disk. When adopting a parametric dust settling prescription, we find that the observations are consistent with a turbulent viscosity coefficient of about α ≲ 10 ⁻⁵ at 100 au. Finally, we find that the thin dust scale height measured in Oph 163131 is favorable for planetary growth by pebble accretion: a 10 M E planet may grow within less than 10 Myr, even in orbits exceeding 50 au. A likely flyby of binary protostar Z CMa caught in action Article Full-text available Mar 2022 Nat. Astron. <here is a image 415a541fcecfd374-1118973e3a10a12a> Hauyu Baobab Liu Motohide Tamura <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Ruobing Dong <here is a image 795827a1436197c2-423b53ef55bc4912> Nicolas Cuello Close encounters between young stellar objects in star-forming clusters are expected to markedly perturb circumstellar disks. Such events are witnessed in numerical simulations of star formation1,2,3, but few direct observations of ongoing encounters have been made. Here we report sub-0.1″-resolution Atacama Large Millimeter/Submillimeter Array and Jansky Very Large Array observations towards the million-year-old binary protostar Z Canis Majoris in dust continuum and molecular line emission. A point source ~4,700 au from the binary has been discovered at both millimetre and centimetre wavelengths. It is located along the extension of a ~2,000 au streamer structure previously found in scattered light imaging, whose counterpart in dust and gas emission is also newly identified. Comparison with simulations shows signposts of a rare flyby event in action. Z CMa is a ‘double burster’, as both binary components undergo accretion outbursts⁴, which may be facilitated by perturbations to the host disk by flybys5,6,7,8. The protoplanetary disc around HD 169142: circumstellar or circumbinary? Article Full-text available Dec 2021 MON NOT R ASTRON SOC D. J. Price Pedro P. Poblete <here is a image 795827a1436197c2-423b53ef55bc4912> Nicolas Cuello <here is a image 01d52cef581ccf05-adba71de9fce25d8> Sebastian Perez Stellar binaries represent a substantial fraction of stellar systems, especially among young stellar objects. Accordingly, binaries play an important role in setting the architecture of a large number of protoplanetary discs. Binaries in coplanar and polar orientations with respect to the circumbinary disc are stable configurations and could induce non-axisymmetric structures in the dust and gas distributions. In this work, we suggest that the structures shown in the central region of the protoplanetary disc HD 169142 are produced by the presence of an inner stellar binary and a circumbinary (P-type) planet. We find that a companion with a mass-ratio of 0.1, semi-major axis of 9.9 au, eccentricity of 0.2, and inclination of 90○, together with a 2 MJ coplanar planet on a circular orbit at 45 au reproduce the structures at the innermost ring observed at 1.3 mm and the shape of spiral features in scattered light observations. The model predicts changes in the disc’s dust structure, and star’s astrometric parameters, which would allow testing its veracity by monitoring this system over the next 20 years. Dust growth, fragmentation, and self-induced dust traps in PHANTOM Article Full-text available Aug 2021 MON NOT R ASTRON SOC <here is a image 3f95155ef30c64d1-219c27ab1e94a0f6> Arnaud Vericel <here is a image 223098871ed766d4-021f9719690ba9ea> Jean-François Gonzalez Daniel J. Price Christophe Pinte We present the implementation of a dust growth and fragmentation module in the public Smoothed Particle Hydrodynamics (SPH) code Phantom. This module is made available for public use with this paper. The coagulation model considers locally monodisperse dust size distributions around single values that are carried by the SPH particles. Along with the presentation of the model, implementation and tests, we showcase growth and fragmentation in a few typical circumstellar disc simulations and revisit previous results. The module is also interfaced with the radiative transfer code mcfost, which facilitates the comparison between simulations and ALMA observations by generating synthetic maps. Circumstellar disc simulations with growth and fragmentation reproduce the ‘self-induced dust trap’ mechanism first proposed in 2017, which supports its existence. Synthetic images of discs featuring this mechanism suggest it would be detectable by ALMA as a bright axisymmetric ring at several tens of au from the star. With this paper, our aim is to provide a public tool to be able to study and explore dust growth in a variety of applications related to planet formation. Spirals, shadows, and precession in HD 100453 – I. The orbit of the binary Article Full-text available Oct 2020 MON NOT R ASTRON SOC <here is a image 223098871ed766d4-021f9719690ba9ea> Jean-François Gonzalez G. van der Plas <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Anne-Lise Maire Christophe Pinte In recent years, several protoplanetary discs have been observed to exhibit spirals, both in scattered light and (sub)millimetre continuum data. The HD 100453 binary star system hosts such a disc around its primary. Previous work has argued that the spirals were caused by the gravitational interaction of the secondary, which was assumed to be on a circular orbit, coplanar with the disc (meaning here the large outer disc, as opposed to the very small inner disc). However, recent observations of the CO gas emission were found incompatible with this assumption. In this paper, we run SPH simulations of the gas and dust disc for seven orbital configurations taken from astrometric fits and compute synthetic observations from their results. Comparing to high-resolution ALMA 12CO data, we find that the best agreement is obtained for an orbit with eccentricity e = 0.32 and semimajor axis a = 207 au, inclined by 61° relative to the disc plane. The large misalignment between the disc and orbit planes is compatible with the tidal evolution of a circumprimary disc in an eccentric, unequal-mass binary star. Spiral arms in the protoplanetary disc HD100453 detected with ALMA: evidence for binary–disc interaction and a vertical temperature gradient Article Full-text available Jan 2020 MON NOT R ASTRON SOC <here is a image 333b91953e03ca2f-cb1bb5aa3f4c034c> Giovanni Rosotti M. Benisty <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> A. Juhász <here is a image 63e4cdacdbcd8408-c7d482c08843efbe> Tomas Stolker Scattered light high-resolution imaging of the protoplanetary disc orbiting HD100453 shows two symmetric spiral arms, possibly launched by an external stellar companion. In this paper, we present new, sensitive high-resolution (∼30 mas) Band 7 ALMA observations of this source. This is the first source where we find counterparts in the sub-mm continuum to both scattered light spirals. The CO J = 3–2 emission line also shows two spiral arms; in this case, they can be traced over a more extended radial range, indicating that the southern spiral arm connects to the companion position. This is clear evidence that the companion is responsible for launching the spirals. The pitch angle of the submillimetre continuum spirals (∼6°) is lower than the one in scattered light (∼16°). We show that hydrodynamical simulations of binary–disc interaction can account for the difference in pitch angle only if one takes into account that the mid-plane is colder than the upper layers of the disc, as expected for the case of externally irradiated discs. External photoevaporation of circumstellar discs constrains the time-scale for planet formation Article Full-text available Dec 2019 MON NOT R ASTRON SOC Francisca Concha-Ramírez Martijn J. C. Wilhelm Simon Portegies Zwart <here is a image 86e230e7b2a5fd0a-ca578f1b4d7ff62f> Thomas James Haworth Planet-forming circumstellar discs are a fundamental part of the star formation process. Since stars form in a hierarchical fashion in groups of up to hundreds or thousands, the UV radiation environment that these discs are exposed to can vary in strength by at least six orders of magnitude. This radiation can limit the masses and sizes of the discs. Diversity in star forming environments can have long lasting effects in disc evolution and in the resulting planetary populations. We perform simulations to explore the evolution of circumstellar discs in young star clusters. We include viscous evolution, as well as the impact of dynamical encounters and external photoevaporation. We find that photoevaporation is an important process in destroying circumstellar discs: in regions of stellar density ρ ∼ 100 M⊙ pc−3 around $80{{\ \rm per\ cent}}$ of discs are destroyed before $2\, \hbox{Myr}$ of cluster evolution. In regions of ρ ∼ 50 M⊙ pc−3 around $50{{\ \rm per\ cent}}$ of discs are destroyed in the same time-scale. Our findings are in agreement with observed disc fractions in young star-forming regions and support previous estimations that planet formation must start in time-scales <0.1–1 Myr. Ongoing flyby in the young multiple system UX Tauri Article Full-text available Jun 2020 F. Menard <here is a image 795827a1436197c2-423b53ef55bc4912> Nicolas Cuello C. Pinte C. Ginski We present observations of the young multiple system UX Tauri to look for circumstellar disks and for signs of dynamical interactions. We obtained SPHERE/IRDIS deep differential polarization images in the J and H bands. We also used ALMA archival CO data. Large extended spirals are well detected in scattered light coming out of the disk of UX Tau A. The southern spiral forms a bridge between UX Tau A and C. These spirals, including the bridge connecting the two stars, all have a CO (3–2) counterpart seen by ALMA. The disk of UX Tau C is detected in scattered light. It is much smaller than the disk of UX Tau A and has a major axis along a different position angle, suggesting a misalignment. We performed PHANTOM SPH hydrodynamical models to interpret the data. The scattered light spirals, CO emission spirals and velocity patterns of the rotating disks, and the compactness of the disk of UX Tau C all point to a scenario in which UX Tau A has been perturbed very recently (∼1000 years) by the close passage of UX Tau C. Spirals inside the millimeter cavity of transition disk SR 21 Article Full-text available Mar 2020 G. A. Muro-Arena C. Ginski C. Dominik Wilhelm Kley Context. Hydrodynamical simulations of planet-disk interactions suggest that planets may be responsible for a number of the substructures frequently observed in disks in both scattered light and dust thermal emission. Despite the ubiquity of these features, direct evidence of planets embedded in disks and of the specific interaction features like spiral arms within planetary gaps are still rare. Aims. In this study we discuss recent observational results in the context of hydrodynamical simulations in order to infer the properties of a putative embedded planet in the cavity of a transition disk. Methods. We imaged the transition disk SR 21 in H -band in scattered light with SPHERE/IRDIS and in thermal dust emission with ALMA band 3 (3 mm) observations at a spatial resolution of 0.1″. We combine these datasets with existing Band 9 (430 μ m) and Band 7 (870 μ m) ALMA continuum data. Results. The Band 3 continuum data reveals a large cavity and a bright ring peaking at 53 au strongly suggestive of dust trapping. The ring shows a pronounced azimuthal asymmetry, with a bright region in the northwest that we interpret as a dust overdensity. A similarly asymmetric ring is revealed at the same location in polarized scattered light, in addition to a set of bright spirals inside the millimeter cavity and a fainter spiral bridging the gap to the outer ring. These features are consistent with a number of previous hydrodynamical models of planet-disk interactions, and suggest the presence of a ∼1 MJup planet at 44 au and PA = 11 deg. This makes SR21 the first disk showing spiral arms inside the millimeter cavity, and the first disk for which the location of a putative planet can be precisely inferred. Conclusions. The main features of SR 21 in both scattered light and thermal emission are consistent with hydrodynamical predictions of planet-disk interactions. With the location of a possible planet being well constrained by observations, it is an ideal candidate for follow-up observations to search for direct evidence of a planetary companion still embedded in its disk. On the Planetary Interpretation of Multiple Gaps and Rings in Protoplanetary Disks Seen By ALMA Close encounters: How stellar flybys shape planet-forming discs We review the role of stellar flybys and encounters in shaping planet-forming discs around young stars, based on the published literature on this topic in the last 30 years. Since most stars $\le ~2$ Myr old harbour protoplanetary discs, tidal perturbations affect planet formation. First, we examine the probability of experiencing flybys or encounters: More than 50% of stars with planet-forming discs in a typical star-forming environment should experience a close stellar encounter or flyby within 1000 au. Second, we detail the dynamical effects of flybys on planet-forming discs. Prograde, parabolic, disc-penetrating flybys are the most destructive. Grazing and penetrating flybys in particular lead to the capture of disc material by the secondary to form a highly misaligned circumsecondary disc with respect to the disc around the primary. One or both discs may undergo extreme accretion and outburst events, similar to the ones observed in FU Orionis-type stars. Warps and broken discs are distinct signatures of retrograde flybys. Third, we review some recently observed stellar systems with discs where a stellar flyby or an encounter is suspected—including UX Tau, RW Aur, AS 205, Z CMa, and FU Ori. Finally, we discuss the implications of stellar flybys for planet formation and exoplanet demographics, including possible imprints of a flyby in the Solar System in the orbits of trans-Neptunian objects and the Sun’s obliquity. Preferential Accretion and Circumbinary Disk Precession in Eccentric Binary Systems We present a suite of high resolution hydrodynamic simulations of binaries immersed in circumbinary accretion disks (CBDs). For the first time, we investigate the preferential accretion rate as a function of both eccentricity eb and mass ratio qb in a densely sampled parameter space, finding that when compared with circular binaries, (i) mass ratios grow more efficiently in binaries on moderately eccentric orbits (0.0 ≲ eb ≲ 0.4), and (ii) high eccentricities (eb ≳ 0.6) suppress mass ratio growth. We suggest that this non-monotonic preferential accretion behaviour may produce an observable shift in the mass ratio distributions of stellar binaries and massive black hole binaries. We further find that the response of a CBD can be divided into three regimes, depending on eccentricity and mass ratio: (i) CBDs around circular binaries always precess freely, whereas CBDs around eccentric binaries either (ii) undergo forced precession or (iii) remain locked at an angle with respect to the binary periapsis. Forced precession in eccentric binaries is associated with strong modulation of individual accretion rates on the precession timescale, a potentially observable signature in accreting binaries with short orbital periods. We provide CBD locking angles and precession rates as a function of eb and qb for our simulation suite. Sustained FU Orionis-type outbursts from colliding discs in stellar flybys We perform 3D hydrodynamics simulations of disc-disc stellar flybys with on-the-fly Monte Carlo radiative transfer. We show that pre-existing circumstellar discs around both stars result in fast rising (∼yrs) outbursts lasting 2–5 times longer than for a star-disc flyby. The perturber always goes into outburst ($\dot{M}>10^{-5}~{\rm M_{\odot }~ yr^{-1}}$). Whereas we find that the primary goes into a decades long outburst only when the flyby is retrograde to the circumprimary disc rotation. High accretion rates during the outburst are triggered by angular momentum cancellation in misaligned material generated by the encounter. A large fraction of accreted material is alien. Excitation of vertical breathing motion in disc galaxies by tidally-induced spirals in fly-by interactions It is now clear that the stars in the Solar neighbourhood display large-scale coherent vertical breathing motions. At the same time, Milky Way-like galaxies experience tidal interactions with satellites/companions during their evolution. While these tidal interactions can excite vertical oscillations, it is still not clear whether vertical breathing motions are excited directly by the tidal encounters or are driven by the tidally-induced spirals. We test whether excitation of breathing motions are directly linked to tidal interactions by constructing a set of N-body models (with mass ratio 5:1) of unbound, single fly-by interactions with varying orbital configurations. We first reproduce the well-known result that such fly-by interactions can excite strong transient spirals (lasting for $\sim 2.9-4.2 \mbox{$\:{\rm Gyr}$}$) in the outer disc of the host galaxy. The generation and strength of the spirals are shown to vary with the orbital parameters (the angle of interaction, and the orbital spin vector). Furthermore, we demonstrate that our fly-by models exhibit coherent breathing motions whose amplitude increases with height. The amplitudes of breathing motions show characteristic modulation along the azimuthal direction, with compressing breathing motions coinciding with the peaks of the spirals and expanding breathing motions falling in the inter-arm regions – a signature of a spiral-driven breathing motion. These breathing motions in our models end when the strong tidally-induced spiral arms fade away. Thus, it is the tidally-induced spirals which drive the large-scale breathing motions in our fly-by models, and the dynamical role of the tidal interaction in this context is indirect. Accretion on to a binary from a polar circumbinary disc We present hydrodynamical simulations to model the accretion flow from a polar circumbinary disc on to a high eccentricity (e = 0.78) binary star system with near unity mass ratio (q = 0.83), as a model for binary HD 98800 BaBb. We compare the polar circumbinary disc accretion flow with the previously studied coplanar case. In the coplanar case, the circumbinary disc becomes eccentric and the accretion alternates from being dominant on to one binary member to the other. For the polar disc case involving a highly eccentric binary, we find that the circumbinary disc retains its initially low eccentricity and that the primary star accretion rate is always about the same as the secondary star accretion rate. Recent observations of the binary HD 98800 BaBb, which has a polar circumbinary disc, have been used to determine the value of the $\rm H\alpha$ flux from the brighter component. From this value, we infer that the accretion rate is much lower than for typical T Tauri stars. The eccentric orbit of the outer companion HD 98800 A increases the accretion rate on to HD 98800 B by ∼20 per cent after each periastron passage. Our hydrodynamical simulations are unable to explain such a low accretion rate unless the disc viscosity parameter is very small, α < 10−5. Additional observations of this system would be useful to check on this low accretion rate. Planet Formation: Key Mechanisms and Global Models Models of planet formation are built on underlying physical processes. In order to make sense of the origin of the planets we must first understand the origin of their building blocks. This review comes in two parts. The first part presents a detailed description of six key mechanisms of planet formation: The structure and evolution of protoplanetary disks The formation of planetesimals Accretion of protoplanets Orbital migration of growing planets Gas accretion and giant planet migration Resonance trapping during planet migration. While this is not a comprehensive list, it includes processes for which our understanding has changed in recent years or for which key uncertainties remain. The second part of this review shows how global models are built out of planet formation processes. We present global models to explain different populations of known planetary systems, including close-in small/low-mass planets (i.e., super-Earths), giant exoplanets, and the Solar System’s planets. We discuss the different sources of water on rocky exoplanets, and use cosmochemical measurements to constrain the origin of Earth’s water. We point out the successes and failings of different models and how they may be falsified. Finally, we lay out a path for the future trajectory of planet formation studies. A Simple Time-Dependent Method for Calculating Spirals: Applications to Eccentric Planets in Protoplanetary discs Spirals in protoplanetary discs have been used to locate the potential planet in discs. Since only the spiral shape from a circularly orbiting perturber is known, most previous works assume that the planet is in a circular orbit. We develop a simple semi-analytical method to calculate the shape of the spirals launched by an eccentric planet. We assume that the planet emits wavelets during its orbit, and the wave fronts of these propagating wavelets form the spirals. The resulting spiral shape from this simple method agrees with numerical simulations exceptionally well. The spirals excited by an eccentric planet can detach from the planet, bifurcate, or even cross each other, which are all reproduced by this simple method. The spiral’s bifurcation point corresponds to the wavelet that is emitted when the planet’s radial speed reaches the disc’s sound speed. Multiple spirals can be excited by an eccentric planet (more than 5 spirals when e ≳ 0.2). The pitch angle and pattern speed are different between different spirals and can vary significantly across one spiral. The spiral wakes launched by high-mass eccentric planets steepen to spiral shocks and the crossing of spiral shocks leads to distorted or broken spirals. With the same mass, a more eccentric planet launches weaker spirals and induces a shallower gap over a long period of time. The observed unusually large/small pitch angles of some spirals, the irregular multiple spirals, and the different pattern speeds between different spirals may suggest the existence of eccentric perturbers in protoplanetary discs. On the rise times in FU Orionis events We examine whether stellar flyby simulations can initiate FU Orionis outbursts using 3D hydrodynamics simulations coupled to live Monte Carlo radiative transfer. We find that a flyby where the secondary penetrates the circumprimary disc triggers a 1–2 year rise in the mass accretion rate to 10−4 M⊙ yr−1 that remains high (≳ 10−5 M⊙ yr−1) for more than a hundred years, similar to the outburst observed in FU Ori. Importantly, we find that the less massive star becomes the dominant accretor, as observed. Thresholds for Particle Clumping by the Streaming Instability The streaming instability (SI) is a mechanism to aerodynamically concentrate solids in protoplanetary disks and trigger the formation of planetesimals. The SI produces strong particle clumping if the ratio of solid to gas surface density - an effective metallicity - exceeds a critical value. This critical value depends on particle sizes and disk conditions such as radial drift-inducing pressure gradients and levels of turbulence. To quantify these thresholds, we perform a suite of vertically stratified SI simulations over a range of dust sizes and metallicities. We find a critical metallicity as low as 0.4% for the optimum particle sizes and standard radial pressure gradients (normalized value of Π = 0.05). This subsolar metallicity is lower than previous results, due to improved numerical methods and computational effort. We discover a sharp increase in the critical metallicity for small solids, when the dimensionless stopping time (Stokes number) is ≤0.01. We provide simple fits to the size-dependent SI clumping threshold, including generalizations to different disk models and levels of turbulence. We also find that linear, unstratified SI growth rates are a surprisingly poor predictor of particle clumping in nonlinear, stratified simulations, especially when the finite resolution of simulations is considered. Our results widen the parameter space for the SI to trigger planetesimal formation. © 2021. The American Astronomical Society. All rights reserved.. Observational Signatures of Planets in Protoplanetary Disks: Temperature Structures in Spiral Arms A dusty filament and turbulent CO spirals in HD 135344B - SAO 206462 Planet-disc interactions build up local pressure maxima that may halt the radial drift of protoplanetary dust, and pile it up in rings and crescents. ALMA observations of the HD 135344B disc revealed two rings in the thermal continuum stemming from ∼mm-sized dust. At higher frequencies the inner ring is brighter relative to the outer ring, which is also shaped as a crescent rather than a full ring. In near-IR scattered light images, the disc is modulated by a 2-armed grand-design spiral originating inside the ALMA inner ring. Such structures may be induced by a massive companion evacuating the central cavity, and by a giant planet in the gap separating both rings, that channels the accretion of small dust and gas through its filamentary wakes while stopping the larger dust from crossing the gap. Here we present ALMA observations in the J = (2 − 1) CO isotopologue lines and in the adjacent continuum, with up to 12 km baselines. Angular resolutions of ∼0${_{.}^{\prime\prime}}$03 reveal the tentative detection of a filament connecting both rings, and which coincides with a local discontinuity in the pitch angle of the IR spiral, proposed previously as the location of the protoplanet driving this spiral. Line diagnostics suggest that turbulence, or superposed velocity components, is particularly strong in the spirals. The 12CO(2-1) 3-D rotation curve points at stellocentric accretion at radii within the inner dust ring, with a radial velocity of up to $\sim 5\%\pm 0.5\%$ Keplerian, which corresponds to an excessively large accretion rate of ∼2 × 10−6 M⊙ yr−1 if all of the CO layer follows the 12CO(2-1) kinematics. This suggests that only the surface layers of the disc are undergoing accretion, and that the line broadening is due to superposed laminar flows. Streaming instability of multiple particle species. II. Numerical convergence with increasing particle number The streaming instability provides an efficient way of overcoming the growth barriers in the initial stages of the planet formation process. Considering the realistic case of a particle size distribution, the dynamics of the system is altered compared to the outcome of single size models. In order to understand the outcome of the multispecies streaming instability in detail, we perform a large parameter study in terms of particle number, particle size distribution, particle size range, initial metallicity, and initial particle scale height. We study vertically stratified systems and determine the metallicity threshold for filament formation. We compare these with a system where the initial particle distribution is unstratified and find that its evolution follows that of its stratified counterpart. We find that a change in the particle number does not result in significant variation in the efficiency and timing of filament formation. We also see that there is no clear trend for how varying the size distribution in combination with the particle size range changes the outcome of the multispecies streaming instability. Finally, we find that an initial metallicity of Z init = 0.005 and Z init = 0.01 both result in similar critical metallicity values for the start of filament formation. Our results show that the inclusion of a particle size distribution into streaming instability simulations, while changing the dynamics as compared to mono-disperse systems, does not result in overall unfavorable conditions for solid growth. We attribute the subdominant role of multiple species to the high-density conditions in the midplane, conditions under which linear stability analysis also predict little difference between single and multiple species. A Dynamical Measurement of the Disk Mass in Elias 2–27 On the Correlation between Hot Jupiters and Stellar Clustering: High-eccentricity Migration Induced by Stellar Flybys A recent observational study suggests that the occurrence of hot Jupiters (HJs) around solar-type stars is correlated with stellar clustering. We study a new scenario for HJ formation, called “Flyby Induced High-e Migration,” that may help explain this correlation. In this scenario, stellar flybys excite the eccentricity and inclination of an outer companion (giant planet, brown dwarf, or low-mass star) at large distance (10–300 au), which then triggers high-e migration of an inner cold Jupiter (at a few astronomical units) through the combined effects of von Zeipel–Lidov–Kozai (ZLK) eccentricity oscillation and tidal dissipation. Using semianalytical calculations of the effective ZLK inclination window, together with numerical simulations of stellar flybys, we obtain the analytic estimate for the HJ occurrence rate in this formation scenario. We find that this “flyby induced high-e migration” could account for a significant fraction of the observed HJ population, although the result depends on several uncertain parameters, including the density and lifetime of birth stellar clusters, and the occurrence rate of the “cold Jupiter + outer companion” systems. Spiral structures in gravito-turbulent gaseous disks Context. Gravitational instabilities can drive small-scale turbulence and large-scale spiral arms in massive gaseous disks under conditions of slow radiative cooling. These motions affect the observed disk morphology, its mass accretion rate and variability, and could control the process of planet formation via dust grain concentration, processing, and collisional fragmentation. Aims. We study gravito-turbulence and its associated spiral structure in thin gaseous disks subject to a prescribed cooling law. We characterize the morphology, coherence, and propagation of the spirals and examine when the flow deviates from viscous disk models. Methods. We used the finite-volume code P LUTO to integrate the equations of self-gravitating hydrodynamics in three-dimensional spherical geometry. The gas was cooled over longer-than-orbital timescales to trigger the gravitational instability and sustain turbulence. We ran models for various disk masses and cooling rates. Results. In all cases considered, the turbulent gravitational stress transports angular momentum outward at a rate compatible with viscous disk theory. The dissipation of orbital energy happens via shocks in spiral density wakes, heating the disk back to a marginally stable thermal equilibrium. These wakes drive vertical motions and contribute to mix material from the disk with its corona. They are formed and destroyed intermittently, and they nearly corotate with the gas at every radius. As a consequence, large-scale spiral arms exhibit no long-term global coherence, and energy thermalization is an essentially local process. Conclusions. In the absence of radial substructures or tidal forcing, and provided a local cooling law, gravito-turbulence reduces to a local phenomenon in thin gaseous disks. Particle Dynamics in 3D Self-gravitating Disks. I. Spirals Spiral arms are distinctive features of many circumstellar disks, observed in scattered light, which traces the disk surface; millimeter dust emission, which probes the disk midplane; as well as molecular emission. The two leading explanations for spirals are wakes generated by a massive planet and the density waves excited by disk self-gravity. We use stratified 3D hydrodynamic shearing-box simulations including dust particles and disk self-gravity to investigate how gas and dust spirals in a self-gravitating disk depend on the simulation size, the cooling efficiency, and the aerodynamic properties of particles. We find that the opening angles of spirals are universal (∼10°) and not significantly affected by the size of the computational domain, the cooling time, or the particle size. In simulations with the biggest domain, the spirals in the gaseous disk become slightly more open with a higher cooling efficiency. Small dust follows the gaseous spirals very well, while intermediate-sized dust with dimensionless stopping time (St) close to 1 is more concentrated in the spirals and shows stronger spirals. However, large dust with St > 1 also shows spirals, which is different from some previous simulations. We identify that this is due to the gravity from the gas to the dust component. We show that when St ≳ Q , the gravitational force from the gaseous spirals on the dust particles becomes stronger than the particles’ aerodynamic drag force, so that the gas significantly affects these large particles through gravitational interaction. This has important implications for both spiral observations and planetesimal formation/dynamics. The evolution of large cavities and disc eccentricity in circumbinary discs We study the mutual evolution of the orbital properties of high-mass ratio, circular, co-planar binaries and their surrounding discs, using 3D Smoothed Particle Hydrodynamics simulations. We investigate the evolution of binary and disc eccentricity, cavity structure, and the formation of orbiting azimuthal overdense features in the disc. Even with circular initial conditions, all discs with mass ratios q > 0.05 develop eccentricity. We find that disc eccentricity grows abruptly after a relatively long time-scale (∼400–700 binary orbits), and is associated with a very small increase in the binary eccentricity. When disc eccentricity grows, the cavity semimajor axis reaches values $a_{\rm cav}\approx 3.5\, a_{\rm bin}$. We also find that the disc eccentricity correlates linearly with the cavity size. Viscosity and orbit crossing appear to be responsible for halting the disc eccentricity growth – eccentricity at the cavity edge in the range ecav ∼ 0.05–0.35. Our analysis shows that the current theoretical framework cannot fully explain the origin of these evolutionary features when the binary is almost circular (ebin ≲ 0.01); we speculate about alternative explanations. As previously observed, we find that the disc develops an azimuthal overdense feature in Keplerian motion at the edge of the cavity. A low-contrast overdensity still co-moves with the flow after 2000 binary orbits; such an overdensity can in principle cause significant dust trapping, with important consequences for protoplanetary disc observations. Flybys in protoplanetary discs: I. Gas and dust dynamics We present 3D smoothed particle hydrodynamics simulations of protoplanetary discs undergoing a flyby by a stellar perturber on a parabolic orbit lying in a plane inclined relative to the disc mid-plane. We model the disc as a mixture of gas and dust, with grains ranging from 1 \|$\mu$\|m to 10 cm in size. Exploring different orbital inclinations, periastron distances, and mass ratios, we investigate the disc dynamical response during and after the flyby. We find that flybys induce evolving spiral structure in both gas and dust that can persist for thousands of years after periastron. Gas and dust structures induced by the flyby differ because of drag-induced effects on the dust grains. Variations in the accretion rate by up to an order of magnitude occur over a time-scale of the order of 10 yr or less, inducing FU Orionis-like outbursts. The remnant discs are truncated and warped. The dust disc is left more compact than the gas disc, both because of disc truncation and accelerated radial drift of grains induced by the flyby. Inclination Excitation of Solar System Debris Disk Due to Stellar Flybys Nathaniel W. H. Moore Gongjie Li Fred C. Adams On the survivability of planets in young massive clusters and its implication of planet orbital architectures in globular clusters MON NOT R ASTRON SOC <here is a image 9af2c9be40c94ef7-5ab0df10093f4f59> Maxwell Xu Cai S. Portegies Zwart M. B. N. Kouwenhoven <here is a image 862e00ae5f7a87ab-5875df58405ad875> Rainer Spurzem As of 2019 August, among the more than 4000 confirmed exoplanets, only one has been detected in a globular cluster (GC) M4. The scarce of exoplanet detections motivates us to employ direct N-body simulations to investigate the dynamical stability of planets in young massive clusters (YMC), which are potentially the progenitors of GCs. In an N = 128 k cluster of virial radius 1.7 pc (comparable to Westerlund-1), our simulations show that most wide-orbit planets (a ≥ 20 au) will be ejected within a time-scale of 10 Myr. Interestingly, more than $70{{\ \rm per\ cent}}$ of planets with a < 5 au survive in the 100 Myr simulations. Ignoring planet–planet scattering and tidal damping, the survivability at t Myr as a function of initial semimajor axis a0 in au in such a YMC can be described as fsurv(a0, t) = −0.33log10(a0)(1 − e−0.0482t) + 1. Upon ejection, about $28.8{{\ \rm per\ cent}}$ of free-floating planets (FFPs) have sufficient speeds to escape from the host cluster at a crossing time-scale. The other FFPs will remain bound to the cluster potential, but the subsequent dynamical evolution of the stellar system can result in the delayed ejection of FFPs from the host cluster. Although a full investigation of planet population in GCs requires extending the simulations to multiGyr, our results suggest that wide-orbit planets and free-floating planets are unlikely to be found in GCs. The evolution of pitch angles of spiral arms MON NOT R ASTRON SOC J. E. Pringle C. L. Dobbs In spiral galaxies, the pitch angle, α, of the spiral arms is often proposed as a discriminator between theories for the formation of the spiral structure. In Lin–Shu density wave theory, α stays constant in time, being simply a property of the underlying galaxy. In other theories (e.g. tidal interaction, and self-gravity), it is expected that the arms wind up in time, so that to a first approximation $\cot \alpha \propto t$. For these theories, it would be expected that a sample of galaxies observed at random times should show a uniform distribution of $\cot \alpha$. We show that a recent set of measurements of spiral pitch angles (Yu & Ho) is broadly consistent with this expectation. Flybys in protoplanetary discs – II. Observational signatures MON NOT R ASTRON SOC <here is a image ee1bdd68a6b91c5b-353d150f03372009> Fabien Louvet Daniel Mentiplay Richard Alexander <here is a image 795827a1436197c2-423b53ef55bc4912> Nicolas Cuello Tidal encounters in star clusters perturb discs around young protostars. In Cuello et al., we detailed the dynamical signatures of a stellar flyby in both gas and dust. Flybys produce warped discs, spirals with evolving pitch angles, increasing accretion rates, and disc truncation. Here, we present the corresponding observational signatures of these features in optical/near-infrared scattered light and (sub) millimetre continuum and CO line emission. Using representative prograde and retrograde encounters for direct comparison, we post-process hydrodynamical simulations with radiative transfer methods to generate a catalogue of multiwavelength observations. This provides a reference to identify flybys in recent near-infrared and submillimetre observations (e.g. RW Aur, AS 205, HV Tau and DO Tau, FU Ori, V2775 Ori, and Z CMa). Flyby encounters between two planetary systems II: exploring the interactions of diverse planetary system architectures MON NOT R ASTRON SOC Daohai Li Alexander J. Mustill Melvyn B. Davies Planetary systems formed in clusters may be subject to stellar encounter flybys. Here, we create a diverse range of representative planetary systems with different orbital scales and planets’ masses and examine encounters between them in a typical open cluster. We first explore the close-in multisuper Earth systems ≲0.1 au. They are resistant to flybys in that only ones inside a few au can destabilize a planet or break the resonance between such planets. But these systems may capture giant planets on to wide orbits from the intruding star during distant flybys. If so, the original close-in small planets’ orbits may be tilted together through Kozai–Lidov mechanism, forming a ‘cold’ system that is significantly inclined against the equator of the central host. Moving to the intermediately placed planets around solar-like stars, we find that the planets’ mass gradient governs the systems’ long-term evolution post-encounter: more massive planets have better chances to survive. Also, a system’s angular momentum deficit, a quantity describing how eccentric/inclined the orbits are, measured immediately after the encounter, closely relates to the longevity of the systems – whether or not and when the systems turn unstable in the ensuing evolution millions of years post-encounter. We compare the orbits of the surviving planets in the unstable systems through (1) the immediate consequence of the stellar fly or (2) internal interplanetary scattering long post-encounter and find that those for the former are systematically colder. Finally, we show that massive wide-orbit multiplanet systems like that of HR 8799 can be easily disrupted and encounters at a few hundreds of au suffice. Binary-induced spiral arms inside the disc cavity of AB Aurigae MON NOT R ASTRON SOC Pedro P. Poblete <here is a image b05f439053949c66-6c6083592448ef1e> Josh Calcino Christophe Pinte <here is a image 795827a1436197c2-423b53ef55bc4912> Nicolas Cuello In this work we demonstrate that the inner spiral structure observed in AB Aurigae can be created by a binary star orbiting inside the dust cavity. We find that a companion with a mass-ratio of 0.25, semimajor axis of 40 au, eccentricity of 0.5, and inclination of 90° produces gaseous spirals closely matching the ones observed in 12CO (2-1) line emission. Based on dust dynamics in circumbinary discs (Poblete, Cuello & Cuadra 2019), we constrain the inclination of the binary with respect to the circumbinary disc to range between 60° and 90°. We predict that the stellar companion is located roughly 0.18 arcsec from the central star towards the east-southeast, above the plane of the disc. Should this companion be detected in the near future, our model indicates that it should be moving away from the primary star at a rate of 6 mas yr−1 on the plane of the sky. Since our companion is inclined, we also predict that the spiral structure will appear to change with time, and not simply corotate with the companion. A Terrestrial-mass Rogue Planet Candidate Detected in the Shortest-timescale Microlensing Event Przemek Mróz Radosław Poleski Andrew Gould Richard W. Pogge Some low-mass planets are expected to be ejected from their parent planetary systems during early stages of planetary system formation. According to planet-formation theories, such as the core accretion theory, typical masses of ejected planets should be between 0.3 and 1.0 M_⊕. Although in practice such objects do not emit any light, they may be detected using gravitational microlensing via their light-bending gravity. Microlensing events due to terrestrial-mass rogue planets are expected to have extremely small angular Einstein radii (< 1 uas) and extremely short timescales (< 0.1 day). Here, we present the discovery of the shortest-timescale microlensing event, OGLE-2016-BLG-1928, identified to date (t_E ≈ 0.0288 day = 41.5min). Thanks to the detection of finite-source effects in the light curve of the event, we were able to measure the angular Einstein radius of the lens θ_E = 0.842±0.064 uas, making the event the most extreme short-timescale microlens discovered to date. Depending on its unknown distance, the lens may be a Mars- to Earth-mass object, with the former possibility favored by the Gaia proper motion measurement of the source. We rule out stellar companions up to the projected distance of 8.0 au from the planet. Our discovery demonstrates that terrestrial-mass free-floating planets can be detected and characterized using microlensing. Planetary Architectures in Interacting Stellar Environments Article Jun 2020 <here is a image 1b40e95fa372219e-886886526863e25d> Yihan Wang Rosalba Perna Nathan W. C. Leigh The discovery of exoplanetary systems has challenged some of the theories of planet formation, which assume unperturbed evolution of the host star and its planets. However, in star clusters the interactions with fly-by stars and binaries may be relatively common during the lifetime of a planetary system. Here, via high-resolution N-body simulations of star–planet systems perturbed by interlopers (stars and binaries), we explore the reconfiguration to the planetary system due to the encounters. In particular, via an exploration focused on the strong scattering regime, we derive the fraction of encounters that result in planet ejections, planet transfers, and collisions by the interloper star/binary, as a function of the characteristics of the environment (density, velocity dispersion), and for different masses of the fly-by star/binary. We find that binary interlopers can significantly increase the cross-section of planet ejections and collisions, while they only slightly change the cross-section for planet transfers. Therefore, in environments with high binary fractions, floating planets are expected to be relatively common, while in environments with low binary fractions, where the cross-sections of planet ejection and transfer are comparable, the rate of planet exchanges between two stars will be comparable to the rate of production of free-floating planets. The effects of disc self-gravity and radiative cooling on the formation of gaps and spirals by young planets MON NOT R ASTRON SOC Shangjia Zhang Zhaohuan Zhu We have carried out 2D hydrodynamical simulations to study the effects of disc self-gravity and radiative cooling on the formation of gaps and spirals. (1) With disc self-gravity included, we find stronger, more tightly wound spirals and deeper gaps in more massive discs. The deeper gaps are due to the larger Angular Momentum Flux (AMF) of the waves excited in more massive discs, as expected from the linear theory. The position of the secondary gap does not change, provided that the disc is not extremely massive (Q ≳ 2). (2) With radiative cooling included, the excited spirals become monotonically more open (less tightly wound) as the disc’s cooling time-scale increases. On the other hand, the amplitude and strength of the spirals decrease when the cooling time increases from a small value to ∼1/Ω, but then the amplitude starts to increase again when the cooling time continues to increase. This indicates that radiative dissipation becomes important for waves with Tcool ∼ 1. Consequently, the induced primary gap is narrower and the secondary gap becomes significantly shallower when the cooling time becomes ∼1/Ω. When the secondary gap is present, the position of it moves to the inner disc from the fast cooling cases to the slow cooling cases. The dependence of gap properties on the cooling time-scale (e.g. in AS 209) provides a new way to constrain the disc optical depth and thus disc surface density. Measuring Turbulent Motion in Planet-forming Disks with ALMA: A Detection around DM Tau and Nondetections around MWC 480 and V4046 Sgr Article ASTROPHYS J Kevin Flaherty A. Meredith Hughes <here is a image e9904b4ca88a4143-b650494933c176df> Jacob B. Simon <here is a image e6b80796769c33b2-609a51b5d5951164> Ágnes Kóspál Most planetary systems—including our own—are born within stellar clusters, where interactions with neighboring stars can help shape the system architecture. This paper develops an orbit-averaged formalism to characterize the cluster's mean-field effects, as well as the physics of long-period stellar encounters. Our secular approach allows for an analytic description of the dynamical consequences of the cluster environment on its constituent planetary systems. We analyze special cases of the resulting Hamiltonian, corresponding to eccentricity evolution driven by planar encounters, as well as hyperbolic perturbations upon dissipative disks. We subsequently apply our results to the early evolution of our solar system, where the cluster's collective potential perturbs the solar system's plane, and stellar encounters act to increase the velocity dispersion of the Kuiper Belt. Our results are twofold. First, we find that cluster effects can alter the mean plane of the solar system by ≾1° and are thus insufficient to explain the ψ ≈ 6° obliquity of the Sun. Second, we delineate the extent to which stellar flybys excite the orbital dispersion of the cold classical Kuiper Belt and show that while stellar flybys may grow the cold belt's inclination by the observed amount, the resulting distribution is incompatible with the data. Correspondingly, our calculations place an upper limit on the product of the stellar number density and residence time of the Sun in its birth cluster, η τ ≾ 2 × 10⁴ Myr pc⁻³. The origin of tail-like structures around protoplanetary disks Disks around T Tauri stars with SPHERE (DARTTS-S) II: Twenty-one new polarimetric images of young stellar disks Context. Near-IR polarimetric images of protoplanetary disks enable us to characterize substructures that might be due to the interaction with (forming) planets. The available census is strongly biased toward massive disks around old stars, however. Aims. The DARTTS program aims at alleviating this bias by imaging a large number of T Tauri stars with diverse properties. Methods. DARTTS-S employs VLT/SPHERE to image the polarized scattered light from disks. In parallel, DARTTS-A provides ALMA images of the same targets for a comparison of different dust components. In this work, we present new SPHERE images of 21 circumstellar disks, which is the largest sample released to date. We also recalculated some relevant stellar and disk properties following Gaia DR2. Results. The targets of this work are significantly younger than those published thus far with polarimetric near-IR (NIR) imaging. Scattered light is unambiguously resolved in 11 targets, and some polarized unresolved signal is detected in 3 additional sources. Some disk substructures are detected. However, the paucity of spirals and shadows from this sample reinforces the trend according to which these NIR features are associated with Herbig stars, either because they are older or more massive. Furthermore, disk rings that are apparent in ALMA observations of some targets do not appear to have corresponding detections with SPHERE. Inner cavities larger than ~15 au are also absent from our images, even though they are expected from the spectral energy distribution. On the other hand, 3 objects show extended filaments at larger scale that are indicative of strong interaction with the surrounding medium. All but one of the undetected disks are best explained by their limited size (≲20 au), and the high occurrence of stellar companions in these sources suggests an important role in limiting the disk size. One undetected disk is massive and very large at millimeter wavelengths, implying that it is self-shadowed in the NIR. Conclusions. This work paves the way toward a more complete and less biased sample of scattered-light observations, which is required to interpret how disk features evolve throughout the disk lifetime. The impact of planet wakes on the location and shape of the water ice line in a protoplanetary disk Context. Planets in accretion disks can excite spiral shocks and if these planets are massive enough, they can even open gaps in their vicinity. Both of these effects can influence the overall thermal structure of the disk. Aims. We model planets of different masses and semimajor axes in disks of various viscosities and accretion rates to examine their impact on disk thermodynamics and to highlight the mutable, non-axisymmetric nature of ice lines in systems with massive planets. Methods. We conducted a parameter study using numerical hydrodynamics simulations where we treated viscous heating, thermal cooling, and stellar irradiation as additional source terms in the energy equation, with some runs including radiative diffusion. Our parameter space consists of a grid containing different combinations of planet and disk parameters. Results. Both gap opening and shock heating can displace the ice line, with the effects amplified for massive planets in optically thick disks. The gap region can split an initially hot ( T > 170 K) disk into a hot inner disk and a hot ring just outside of the planet’s location, while shock heating can reshape the originally axisymmetric ice line into water-poor islands along spirals. We also find that radiative diffusion does not alter the picture significantly in this context. Conclusions. Shock heating and gap opening by a planet can effectively heat up optically thick disks and, in general, they can move or reshape the water ice line. This can affect the gap structure and migration torques. It can also produce azimuthal features that follow the trajectory of spiral arms, creating hot zones which lead to “islands” of vapor and ice around spirals that could affect the accretion or growth of icy aggregates.	https://www.researchgate.net/publication/369231350_Exciting_spiral_arms_in_protoplanetary_discs_from_flybys
Association of Patient Characteristics, Diabetes, BMI, and Obesity With Severe COVID-19 in Metropolitan Detroit, MI \| Clinical Diabetes \| American Diabetes Association Identification of specific risk factors for severe coronavirus disease 2019 (COVID-19) is crucial for prevention of poor outcomes and mortality. This retrospect Association of Patient Characteristics, Diabetes, BMI, and Obesity With Severe COVID-19 in Metropolitan Detroit, MI 1 Department of Internal Medicine, Division of Endocrinology, Diabetes, Bone and Mineral Disorders, Henry Ford Health System, Detroit, MI Erika Todter 2 Department of Public Health Sciences, Henry Ford Health System, Detroit, MI Sharon W. Lahiri 0000-0003-0678-6262 Sharon W. Lahiri 1 Department of Internal Medicine, Division of Endocrinology, Diabetes, Bone and Mineral Disorders, Henry Ford Health System, Detroit, MI Corresponding author: Sharon W. Lahiri, slahiri1@hfhs.org Corresponding author: Sharon W. Lahiri, slahiri1@hfhs.org Clin Diabetes 2022;40(2):141–152 https://doi.org/10.2337/cd21-0065 PubMed: 35669303 Identification of specific risk factors for severe coronavirus disease 2019 (COVID-19) is crucial for prevention of poor outcomes and mortality. This retrospective cohort study of patients hospitalized with COVID-19 demonstrated that older age, male sex, Black race, diabetes, elevated BMI, and elevated inflammatory markers were correlated with critical illness in COVID-19. Older age, male sex, diabetes, and inflammatory markers, but not elevated BMI, were associated with mortality. Despite having greater odds of critical illness, Black patients had lower odds of death than White patients. Older age, male sex, diabetes, and elevated inflammatory markers were significantly associated with venous thromboembolism. These findings suggest a need to aggressively identify and manage modifiable risk factors (i.e., diabetes and elevated BMI) and encourage vaccination of at-risk individuals to prevent poor outcomes from COVID-19. The coronavirus disease 2019 (COVID-19) pandemic has tragically claimed millions of lives, profoundly affected well-being, and resulted in dramatic social and economic disruption. The state of Michigan has been deeply affected by COVID-19, having led the nation in the number of new infections and being the only state categorized as at a “severe” risk level in April 2021 (1). The estimated total cost from COVID-19 is >$16 trillion, with approximately half of that cost resulting from lost income from the COVID-19–induced recession and the rest from loss of health (2). Although >94% of individuals survive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (3), severe disease and death can occur, especially in individuals who are older, male, and have comorbid conditions (4). Furthermore, persistent “long-haul” symptoms have been reported among COVID-19 survivors, even in those who experienced an initial mild acute illness (5). The high likelihood of SARS-CoV-2 becoming endemic, lack of a cure, and possibility of severe disease, death, and long-term morbidity from illness reinforce the need to identify risk factors for poor outcomes from COVID-19. The Henry Ford Health System (HFHS), a comprehensive, integrated, nonprofit health care organization that includes five hospitals in metropolitan Detroit, MI, provides a unique patient population to study risk factors for severe COVID-19. A significant portion of patients seen at HFHS comes from Detroit, which is the location of the 877-bed flagship hospital. The aims of our study were to determine the incidence of severe outcomes (i.e., need for mechanical ventilation [MV], intensive care unit [ICU] admission, death, and venous thromboembolism [VTE]) in patients admitted for COVID-19 within a single health system, describe the clinical characteristics of these patients, and evaluate the correlation of poor outcomes with demographics, diabetes, BMI, obesity, and levels of inflammatory markers. Additionally, to identify potential risk factors that are unique to our patient population, we compared patient characteristics and the presence of multiple comorbidities in Black and White patients with COVID-19. Determination of risk factors associated with severe COVID-19, especially in a unique population with a large percentage of Black patients, can guide risk mitigation efforts and help to identify racial gaps in health care. Research Design and Methods Study Design and Participants This was a retrospective study of electronic medical records (EMRs) of adult patients (≥18 years of age) who were admitted to any of the five hospitals in the HFHS (Henry Ford Hospital in Detroit, Henry Ford West Bloomfield Hospital, Henry Ford Macomb Hospital, Henry Ford Wyandotte Hospital, and Henry Ford Allegiance Health in Jackson, MI) and were diagnosed with COVID-19 by real-time PCR between 1 March and 25 July 2020. Polymerase chain reaction testing was validated and performed on four platforms (NeumoDx 288, Cepheid GeneXpert and Infinity, Hologic Panther, and Diasorin Liaison MDX). This study was approved by the HFHS’s institutional review board. The need for informed consent was waived because this was a retrospective chart review with minimal risk and no intervention. Data Collection Demographic, clinical, laboratory, and outcome data were extracted from EMRs. These included age, sex, race (described as Black or White in the EMRs), BMI, A1C, and the presence of comorbidities, including diabetes, hypertension, chronic kidney disease (CKD), sleep apnea, chronic obstructive pulmonary disease (COPD), coronary artery disease (CAD), stroke, heart failure (HF), cirrhosis, HIV infection, and active malignancy. Additionally, the inflammatory markers d-dimer, ferritin, C-reactive protein (CRP), interleukin 6 (IL-6), and procalcitonin were collected. Primary outcomes evaluated were the need for MV, need for ICU admission, death, and development of VTE. Statistical Analysis Descriptive statistics were used to describe patient demographics, clinical characteristics, and multiple outcomes. Obese, overweight, and at least overweight were defined as BMI ≥30.0, 25.0–29.99, and ≥25.0 kg/m 2, respectively. We compared the incidence of comorbid conditions in Black and White patients who had confirmed COVID-19. The average value was used for cases in which patients had multiple BMI or A1C values during their admission. Two-group comparisons using χ 2tests and Fisher’s exact test (when counts were <5) assessed correlation of race with comorbidities and outcomes. Multivariate logistic regression models were used to determine whether demographics, A1C >5.7%, diabetes, elevated BMI categories, or inflammatory markers ( d-dimer, ferritin, CRP, IL-6, and procalcitonin) were significantly associated with the four outcomes. Each analysis controlled for the confounders of age, sex, race, diabetes, BMI, and eight additional comorbidities (hypertension, CKD, CAD, stroke, HF, COPD, sleep apnea, and active malignancy). The analyses for A1C >5.7% were controlled for all previously mentioned confounding variables except diabetes. For the BMI categories, multivariate logistic regression models were used to measure the effects of BMI ≥30.0, ≥25.0, and ≥30.0 vs. 25.0–29.99 kg/m 2on each outcome. In cases with multiple values for the same inflammatory marker during the hospital stay, the earliest value was used. If the inflammatory marker was measured multiple times on the earliest day, then the average value on the earliest day was used. Statistical significance was determined if P<0.05. All analyses were performed using SAS 9.4 software (SAS Institute, Cary, NC). Results Overall Characteristics and Main Outcomes A total of 8,751 adults hospitalized with COVID-19 between 1 March and 25 July 2020 were included in this cohort study, of whom 682 (7.8%) required MV, 867 (9.9%) were admitted to the ICU, 753 (8.6%) died, and 430 (4.9%) were diagnosed with VTE. Of these patients, 4,447 (50.8%) were Black, 4,951 (56.6%) were women, and 5,152 (58.9%) were >50 years of age. Of the patients who had BMI recorded, 2,560 (50.3%) had a BMI ≥30.0 kg/m 2, 1,397 (27.5%) had a BMI of 25.0–29.99 kg/m 2, and 3,957 (77.8%) had a BMI ≥25.0 kg/m 2. Of the patients who had A1C checked, 1,138 (74.3%) had an A1C >5.7%. Diabetes and hypertension were the most frequent comorbidities (2,068 [23.6%] had diabetes and 1,164 [13.3%] had hypertension). Fewer than 10% had the following comorbidities: CKD in 400 (4.6%), sleep apnea in 125 (1.4%), COPD in 155 (1.8%), CAD in 321 (3.7%), stroke in 100 (1.1%), HF in 310 (3.5%), and malignancy in 300 (3.4%) patients. Fewer than 1% of patients had cirrhosis ( n= 35 [0.4%]) or HIV diagnosis ( n= 11 [0.1%]). All descriptive data are listed inTable 1. TABLE 1 Patient Characteristics and Main Outcomes of Patients With COVID-19 ( N= 8,751) Demographics Race Black 4,447 (50.82) White 3,048 (34.83) Other 1,256 (14.35) Sex Male 3,800 (43.42) Female 4,951 (56.58) Age, years 18–29 968 (11.06) 30–39 1,227 (14.02) 40–49 1,404 (16.04) 50–59 1,745 (19.94) 60–69 1,541 (17.61) ≥70 1,866 (21.32) Clinical variables Diabetes 2,068 (23.63) Hypertension 1,164 (13.30) CKD 400 (4.57) Sleep apnea 125 (1.43) COPD 155 (1.77) CAD 321 (3.67) Stroke 100 (1.14) HF 310 (3.54) Cirrhosis 35 (0.40) HIV 11 (0.13) Active malignancy 300 (3.43) Weight status ( n = 5,088) Obesity (BMI ≥30.0 kg/m 2 ) 2,560 (50.31) Overweight (BMI 25.0–29.99 kg/m 2 ) 1,397 (27.46) At least overweight (BMI ≥25.0 kg/m 2 ) 3,957 (77.77) A1C >5.7% ( n = 1,532) 1,138 (74.28) Outcomes MV 682 (7.8) ICU admission 867 (9.9) VTE 430 (4.9) Death 753 (8.6) Demographics Race Black 4,447 (50.82) White 3,048 (34.83) Other 1,256 (14.35) Sex Male 3,800 (43.42) Female 4,951 (56.58) Age, years 18–29 968 (11.06) 30–39 1,227 (14.02) 40–49 1,404 (16.04) 50–59 1,745 (19.94) 60–69 1,541 (17.61) ≥70 1,866 (21.32) Clinical variables Diabetes 2,068 (23.63) Hypertension 1,164 (13.30) CKD 400 (4.57) Sleep apnea 125 (1.43) COPD 155 (1.77) CAD 321 (3.67) Stroke 100 (1.14) HF 310 (3.54) Cirrhosis 35 (0.40) HIV 11 (0.13) Active malignancy 300 (3.43) Weight status ( n = 5,088) Obesity (BMI ≥30.0 kg/m 2 ) 2,560 (50.31) Overweight (BMI 25.0–29.99 kg/m 2 ) 1,397 (27.46) At least overweight (BMI ≥25.0 kg/m 2 ) 3,957 (77.77) A1C >5.7% ( n = 1,532) 1,138 (74.28) Outcomes MV 682 (7.8) ICU admission 867 (9.9) VTE 430 (4.9) Death 753 (8.6) Data are n(%). Association of Demographics and Comorbidities With Race Our data suggested a significant association between race (categorized as Black or White) and having multiple comorbid conditions (diabetes, hypertension, CKD, sleep apnea, COPD, CAD, and cirrhosis), an A1C >5.7%, a BMI ≥25.0 kg/m 2, and a BMI ≥30.0 kg/m 2in patients with COVID-19. Comparison of Black and White patients revealed that significantly greater percentages of Black patients had a BMI ≥25.0 kg/m 2(81.9 vs. 71.2%, P<0.001), a BMI ≥30.0 kg/m 2(57.7 vs. 41.5%, P<0.001), an A1C >5.7% (75.7 vs. 69.7%, P= 0.022), diabetes (27.7 vs. 19.9%, P<0.001), hypertension (17.7 vs. 9.5%, P<0.001), CKD (6.2 vs. 3.4%, P<0.001), and sleep apnea (2.1 vs. 0.7%, P<0.001). The data also suggested that a smaller proportion of Black patients in this population had COPD, CAD, and cirrhosis compared with White patients. Associations of demographics and comorbidities with race are shown inTable 2. TABLE 2 Associations of Demographics and Comorbidities With Race in Patients With COVID-19 ( N= 7,495) . Black ( n = 4,447) . White ( n = 3,048) . P * . Age >50 years 2,556 (57.5) 1,846 (60.6) 0.008 Male sex 1,824 (41.0) 1,369 (44.9) <0.001 Diabetes 1,233 (27.7) 606 (19.9) <0.001 Hypertension 786 (17.7) 291 (9.5) <0.001 CKD 275 (6.2) 103 (3.4) <0.001 Sleep apnea 95 (2.1) 21 (0.7) <0.001 COPD 75 (1.7) 75 (2.5) 0.019 CAD 146 (3.3) 152 (5.0) <0.001 Stroke 58 (1.3) 36 (1.2) 0.638 HF 179 (4.0) 118 (3.9) 0.737 Cirrhosis 10 (0.2) 20 (0.7) 0.004 HIV 9 (0.2) 2 (0.1) 0.218 Active malignancy 167 (3.8) 121 (4.0) 0.635 ( n = 2,690) ( n = 1,779) At least overweight (BMI ≥25.0 kg/m 2 ) 2,202 (81.9) 1,267 (71.2) <0.001 Obese (BMI ≥30.0 kg/m 2 ) 1,553 (57.7) 739 (41.5) <0.001 ( n = 950) ( n = 399) A1C >5.7% 719 (75.7) 278 (69.7) 0.022 . Black ( n = 4,447) . White ( n = 3,048) . P * . Age >50 years 2,556 (57.5) 1,846 (60.6) 0.008 Male sex 1,824 (41.0) 1,369 (44.9) <0.001 Diabetes 1,233 (27.7) 606 (19.9) <0.001 Hypertension 786 (17.7) 291 (9.5) <0.001 CKD 275 (6.2) 103 (3.4) <0.001 Sleep apnea 95 (2.1) 21 (0.7) <0.001 COPD 75 (1.7) 75 (2.5) 0.019 CAD 146 (3.3) 152 (5.0) <0.001 Stroke 58 (1.3) 36 (1.2) 0.638 HF 179 (4.0) 118 (3.9) 0.737 Cirrhosis 10 (0.2) 20 (0.7) 0.004 HIV 9 (0.2) 2 (0.1) 0.218 Active malignancy 167 (3.8) 121 (4.0) 0.635 ( n = 2,690) ( n = 1,779) At least overweight (BMI ≥25.0 kg/m 2 ) 2,202 (81.9) 1,267 (71.2) <0.001 Obese (BMI ≥30.0 kg/m 2 ) 1,553 (57.7) 739 (41.5) <0.001 ( n = 950) ( n = 399) A1C >5.7% 719 (75.7) 278 (69.7) 0.022 Data by racial group are n(%). * Two-group comparisons were performed with χ 2test (or Fisher’s exact test when expected cell counts were <5) to determine associations between race/ethnicity (categorized in EMRs as Black or White) and each variable. Statistical significance is determined if P<0.05. Multivariate Regression Analysis of Patient Characteristics and Serious Outcomes Multivariate logistic regression models were used to determine whether demographic and clinical factors were associated with the four outcomes (MV, ICU admission, death, and VTE). Each analysis was controlled for confounding variables, including age, sex, race, diabetes, BMI, and the eight additional comorbidities listed in S tatisticalA nalysis. Results of multivariate regression analysis are shown inTable 3. TABLE 3 Multivariate Regression Analysis of Characteristics of Patients With COVID-19 and Serious Outcomes Variable . MV . ICU Admission . Death . VTE . OR (95% CI) . P * . OR (95% CI) . P * . OR (95% CI) . P * . OR (95% CI) . P * . Age, years 1.03 (1.03–1.04) <0.0001 1.02 (1.02–1.03) <0.0001 1.08 (1.07–1.09) <0.0001 1.02 (1.01–1.03) <0.0001 Male sex 2.02 (1.70–2.41) <0.0001 1.82 (1.56–2.13) <0.0001 1.82 (1.52–2.19) <0.0001 1.29 (1.04–1.60) 0.0193 Black vs. White race 1.44 (1.13–1.83) 0.0009 1.43 (1.15–1.77) 0.0002 0.77 (0.60–0.97) 0.0200 0.95 (0.71–1.27) >0.99 Diabetes 2.00 (1.68–2.38) <0.0001 2.01 (1.72–2.36) <0.0001 1.50 (1.25–1.80) <0.0001 1.47 (1.17–1.84) 0.0009 At least overweight (BMI ≥25.0 kg/m 2 ) 1.84 (1.46–2.32) <0.0001 1.34 (1.10–1.63) 0.0036 0.90 (0.74–1.11) 0.3205 0.91 (0.70–1.17) 0.4450 Obese (BMI ≥30.0 kg/m 2 ) 1.55 (1.29–1.86) <0.0001 1.40 (1.19–1.65) <0.0001 1.09 (0.90–1.32) 0.4010 1.13 (0.90–1.41) 0.3051 Obese (BMI ≥30.0 kg/m 2 ) vs. overweight (BMI 25.0–29.99 kg/m 2 ) † 1.31 (1.07–1.61) 0.0096 1.36 (1.12–1.64) 0.0017 1.17 (0.93–1.46) 0.1843 1.24 (0.94–1.62) 0.127 Variable . MV . ICU Admission . Death . VTE . OR (95% CI) . P * . OR (95% CI) . P * . OR (95% CI) . P * . OR (95% CI) . P * . Age, years 1.03 (1.03–1.04) <0.0001 1.02 (1.02–1.03) <0.0001 1.08 (1.07–1.09) <0.0001 1.02 (1.01–1.03) <0.0001 Male sex 2.02 (1.70–2.41) <0.0001 1.82 (1.56–2.13) <0.0001 1.82 (1.52–2.19) <0.0001 1.29 (1.04–1.60) 0.0193 Black vs. White race 1.44 (1.13–1.83) 0.0009 1.43 (1.15–1.77) 0.0002 0.77 (0.60–0.97) 0.0200 0.95 (0.71–1.27) >0.99 Diabetes 2.00 (1.68–2.38) <0.0001 2.01 (1.72–2.36) <0.0001 1.50 (1.25–1.80) <0.0001 1.47 (1.17–1.84) 0.0009 At least overweight (BMI ≥25.0 kg/m 2 ) 1.84 (1.46–2.32) <0.0001 1.34 (1.10–1.63) 0.0036 0.90 (0.74–1.11) 0.3205 0.91 (0.70–1.17) 0.4450 Obese (BMI ≥30.0 kg/m 2 ) 1.55 (1.29–1.86) <0.0001 1.40 (1.19–1.65) <0.0001 1.09 (0.90–1.32) 0.4010 1.13 (0.90–1.41) 0.3051 Obese (BMI ≥30.0 kg/m 2 ) vs. overweight (BMI 25.0–29.99 kg/m 2 ) † 1.31 (1.07–1.61) 0.0096 1.36 (1.12–1.64) 0.0017 1.17 (0.93–1.46) 0.1843 1.24 (0.94–1.62) 0.127 * Multivariate logistic regression controlled for age, sex, race, diabetes, BMI, hypertension, CKD, CAD, stroke, HF, COPD, sleep apnea, and active malignancy. Statistical significance was determined if P<0.05. † Results for obese versus overweight represent the odds of the outcome for those who are obese (BMI ≥30.0 kg/m 2) relative to the odds of the outcome for those who are overweight (BMI 25.0–29.99 kg/m 2). Age and male sex were significantly associated with all four outcomes. For age, odds ratios (ORs) indicated that for every 1-year increase in age, the odds of needing MV rose by a factor of 1.03 (95% CI 1.03–1.04, P<0.0001), being admitted to the ICU rose by a factor of 1.02 (95% CI 1.02–1.03, P<0.0001), dying rose by a factor of 1.08 (95% CI 1.07–1.09, P<0.0001), and developing VTE rose by a factor of 1.02 (95% CI 1.01–1.03; P<0.0001). Men were more likely to require MV (OR 2.02, 95% CI 1.70–2.41, P<0.0001), be admitted to the ICU (OR 1.82, 95% CI 1.56–2.13, P<0.0001), die (OR 1.82, 95% CI 1.52–2.19, P<0.0001), and develop VTE (OR 1.29, 95% CI 1.04–1.60, P= 0.0193). Black patients had significantly greater odds of MV (OR 1.44, 95% CI 1.13–1.83, P= 0.0009) and ICU admission (OR 1.43, 95% CI 1.15–1.77, P= 0.0002), but not VTE, than White patients. Black patients had significantly lower odds of death than White patients (OR 0.77, 95% CI 0.60–0.97, P= 0.0200). Diabetes was significantly associated with all four outcomes. The presence of diabetes was associated with MV (OR 2.0, 95% CI 1.68–2.38, P<0.0001), ICU admission (OR 2.01, 95% CI 1.72–2.36, P<0.0001), death (OR 1.50, 95% CI 1.25–1.80, P<0.0001), and VTE (OR 1.47, 95% CI 1.17–1.84, P= 0.0009). A1C >5.7% was also significantly associated with MV (OR 1.97, 95% CI 1.28–3.05, P= 0.0022) and ICU admission (OR 2.11, 95% CI 1.44–3.07, P= 0.0001) but not death or VTE. Weight categories of being at least overweight (BMI ≥25.0 kg/m 2), obese (BMI ≥30.0 kg/m 2), and obese relative to overweight (BMI 25.0–29.99 kg/m 2) status were significantly associated with MV (OR 1.84, 95% CI 1.46–2.32, P<0.0001; OR 1.55, 95% CI 1.29–1.86, P<0.0001; and OR 1.31, 95% CI 1.07–1.61, P= 0.0096, respectively) and ICU admission (OR 1.34, 95% CI 1.10–1.63, P= 0.0036; OR 1.40, 95% CI 1.19–1.65, P<0.0001; and OR 1.36, 95% CI 1.12–1.64, P= 0.0017, respectively). However, none of the weight categories were associated with death or VTE. Multivariate Regression Analysis of Inflammatory Markers and Serious Outcomes Multivariate logistic regression models analyzed the presence of various inflammatory markers on the four outcomes in all patients, controlling for age, sex, race, diabetes, BMI, and the eight additional comorbidities listed in S tatisticalA nalysis. Results of the multivariate logistic regression are inTable 4. TABLE 4 Multivariate Regression Analysis of Inflammatory Markers in Patients With COVID-19 and Serious Outcomes Outcome . Inflammatory Marker . Unit Change . OR (95% CI) . P . MV d -Dimer 1 1.06 (1.04–1.08) <0.0001 Ferritin 100 1.02 (1.01–1.02) <0.0001 CRP 1 1.07 (1.06–1.08) <0.0001 IL-6 10 1.02 (1.01–1.03) 0.0021 Procalcitonin 1 1.03 (1.01–1.04) <0.0001 ICU admission d -Dimer 1 1.07 (1.05–1.08) <0.0001 Ferritin 100 1.01 (1.01–1.02) <0.0001 CRP 1 1.06 (1.05–1.07) <0.0001 IL-6 10 1.01 (1.00–1.02) 0.2506 Procalcitonin 1 1.02 (1.01–1.03) 0.0003 Death d -Dimer 1 1.06 (1.04–1.07) <0.0001 Ferritin 100 1.01 (1.01–1.02) <0.0001 CRP 1 1.05 (1.04–1.07) <0.0001 IL-6 10 1.01 (1.00–1.02) 0.0067 Procalcitonin 1 1.04 (1.03–1.05) <0.0001 VTE d -Dimer 1 1.10 (1.08–1.12) <0.0001 Ferritin 100 1.00 (0.99–1.01) 0.3518 CRP 1 1.02 (1.01–1.03) 0.0094 IL-6 10 1.00 (1.00–1.00) 0.7546 Procalcitonin 1 1.01 (1.00–1.03) 0.0201 Outcome . Inflammatory Marker . Unit Change . OR (95% CI) . P . MV d -Dimer 1 1.06 (1.04–1.08) <0.0001 Ferritin 100 1.02 (1.01–1.02) <0.0001 CRP 1 1.07 (1.06–1.08) <0.0001 IL-6 10 1.02 (1.01–1.03) 0.0021 Procalcitonin 1 1.03 (1.01–1.04) <0.0001 ICU admission d -Dimer 1 1.07 (1.05–1.08) <0.0001 Ferritin 100 1.01 (1.01–1.02) <0.0001 CRP 1 1.06 (1.05–1.07) <0.0001 IL-6 10 1.01 (1.00–1.02) 0.2506 Procalcitonin 1 1.02 (1.01–1.03) 0.0003 Death d -Dimer 1 1.06 (1.04–1.07) <0.0001 Ferritin 100 1.01 (1.01–1.02) <0.0001 CRP 1 1.05 (1.04–1.07) <0.0001 IL-6 10 1.01 (1.00–1.02) 0.0067 Procalcitonin 1 1.04 (1.03–1.05) <0.0001 VTE d -Dimer 1 1.10 (1.08–1.12) <0.0001 Ferritin 100 1.00 (0.99–1.01) 0.3518 CRP 1 1.02 (1.01–1.03) 0.0094 IL-6 10 1.00 (1.00–1.00) 0.7546 Procalcitonin 1 1.01 (1.00–1.03) 0.0201 Multivariate logistic regression models evaluated whether inflammatory markers ( d-dimer, ferritin, CRP, IL-6, and procalcitonin) were associated with each outcome while controlling for age, sex, race, diabetes, BMI, hypertension, CKD, CAD, stroke, HF, COPD, sleep apnea, and active malignancy. In cases with multiple values for the same inflammatory marker during the hospital stay, the earliest value was used. If the inflammatory marker was measured multiple times on the earliest day, then the average value on the earliest day was used. Statistical significance was determined if P<0.05. All inflammatory markers ( d-dimer, ferritin, CRP, IL-6, and procalcitonin) were significantly correlated with risk of MV and death in all patients. All inflammatory markers except IL-6 were significantly correlated with ICU admission in all patients. d-dimer, CRP, and procalcitonin were associated with VTE in all patients, whereas IL-6 and ferritin were not. Discussion This retrospective cohort study of patients hospitalized with COVID-19 within a large health system in metropolitan Detroit showed that older age, male sex, Black race, diabetes, elevated BMI, and elevated inflammatory markers were correlated with critical illness in COVID-19. Older age, male sex, diabetes, and inflammatory markers, but not elevated BMI, were associated with mortality. Despite having greater odds of critical illness, Black patients had lower odds of death than White patients. Older age, male sex, diabetes, and elevated inflammatory markers were significantly associated with VTE. Incidence of Poor Outcomes From COVID-19 Fewer than 10% of our study population was found to require MV or ICU admission, to be diagnosed with VTE, or to die. In contrast, the Centers for Disease Control and Prevention (CDC) estimated that 12% (age <50 years) to 21.6% (age >50 years) of patients with COVID-19 needed MV, 23.8% (age <50 years) to 35.7% (age >50 years) required ICU admission, and 2.4% (age <50 years) to 18.3% (age >50 years) died (3). Overall, VTE incidence is estimated to be 21% (6). The incidence of poor outcomes was lower that CDC estimates in our study, possibly because of earlier intervention with more effective therapies, the large proportion of patients <50 years of age, and effective transition of care from in-person to virtual. Our study included patients who were admitted a few months into the pandemic, when rapidly evolving treatment algorithms started to include regular use of corticosteroids, prone positioning in the ICU, anticoagulation therapy, and use of remdesivir. A large proportion (40%) of our study population was relatively young (<50 years of age), which may partially explain the lower incidence of severe outcomes. HFHS also has a well-established EMR system and had been offering virtual visits before the pandemic, resulting in a smooth and immediate transition to virtual outpatient visits and continued access to health care when the pandemic began, possibly contributing to improved management of comorbid conditions and lower incidence of poor outcomes. Age and Outcomes From COVID-19 CDC data show that the risk of hospitalization and death from COVID-19 increases with age, starting even at 18 years of age, and that a striking increase in risk starts in the 65- to 74-year age range, with the highest risk noted for those ≥85 years of age (up to 8,700-fold higher risk) (7). Our data revealed increased odds of MV and ICU admission, death, and VTE per age year, corroborating the CDC data analysis and findings from studies reporting older age to be associated with higher risk of hospital admission (8,9), higher mortality (10–15), and risk of MV (11,16). Comparisons between studies is difficult because of the heterogeneity of age categories and outcomes studied. Many studies adjusted for some confounders in their analyses, but usually fewer than in our study, perhaps resulting in the lower ORs we observed (1.03–1.08) relative to other studies in similar health care settings with a similar patient population (1.73–5.3) (11,15,17). The only meta-analysis specifically evaluating the effect of age on outcomes included 12 studies, all from China, and emphasized the importance of adjusting for confounders. These authors found significant bias in results and a marked decrease in the influence of age on COVID-19 disease severity and mortality after adjustment for important age-related risk factors (18). We controlled for 13 risk factors and still found a significant association of increasing age with severe disease and mortality. The etiology of an age-related effect on poor outcomes in patients with COVID-19 has been described to be related to increasing baseline inflammation, possibly resulting from increasing levels of senescent cells with age. Senescent cells can activate endothelial cells lining capillaries in the lungs and increase expression of natural killer cell receptor ligands that recruit natural killer-like T cells, which cause collateral lung destruction (19). Male Sex and Outcomes From COVID-19 Male sex has been associated with higher risks of hospital admission and critical illness from COVID-19 (1.3–2 times greater) (8,13,20–22), as well as higher case fatality rate (1.46–1.7 times greater) than for women (16,20–25). Our results similarly found that male sex, controlling for multiple confounders, was significantly associated with all four severe outcomes (ORs 1.29–2.02). Male predominance of deaths was also observed in previous epidemics from SARS-CoV and Middle East respiratory syndrome (26–28). Postulated underlying etiologies include sex-biased expression of the virus entry receptor angiotensin-converting enzyme 2 (ACE2) and regulation of cellular transmembrane serine protease 2 (the enzyme that primes the spike protein of the virus), which could increase the susceptibility of men to SARS-CoV-2 compared with women (25). Furthermore, differences based on sex may exist in innate immunity, possibly related to the location of immune regulatory genes on the second X chromosome and higher CD4+ lymphocytes (and therefore better viral clearance), higher antibody production, higher protective levels of estrogen, and lower production of inflammatory IL-6 in women compared with men (29). Black Race and Outcomes From COVID-19 The literature to date provides strong evidence that Black patients have higher incidences of SARS-CoV-2 infection and hospitalization for COVID-19 compared with White patients. Data on severe outcomes and mortality in Black compared with White patients are less clear; some studies and meta-analyses have found an association of Black race with more severe disease (MV) (14) and COVID-19–related death (13,30–32) compared with White race, whereas other studies have not found any differences (14,33–35). Our study includes a large percentage of Black patients (50%), consistent with the large Black population in Detroit (78.3%) (36). Our results suggested a significant association of Black race with critical illness, but not increased mortality, relative to White patients, adding to the mixed findings in the literature on this topic. The lack of association of Black race with higher mortality is similar to findings from three studies that had similar geographic and racial demographics to our study (56–72% Black patients in the Detroit metropolitan area) (15,17,37). In fact, our data suggested that Black patients had significantly lower odds of death than White patients, similar to results of a smaller study conducted at HFHS by Miller et al. (37). In contrast to other studies, our study also looked at specific differences in the presence of comorbid conditions in Black compared with White patients, finding that Black patients had a significantly higher prevalence of elevated BMI, A1C >5.7%, diabetes, hypertension, CKD, and sleep apnea than White patients. Because we controlled for a variety of comorbidities and other variables (age, sex, race, diabetes, and BMI), the association between Black race and greater odds of critical illness relative to White race may be explained by other variables such as lower socioeconomic status and decreased access to health care or health insurance. We did not evaluate for these factors, but based on 2019 U.S. Census Bureau data for Detroit, 35% of people in this majority Black city are in poverty, and 9.6% of the population do not have health insurance (36), suggesting that socioeconomic hardships are prevalent and likely to contribute to poor health and outcomes. Diabetes and Outcomes From COVID-19 High prevalence rates of diabetes (22–38.4%) have been found in patients hospitalized for COVID-19 in the United States and other countries (8,15,17,37–41), and meta-analyses have demonstrated that the prevalence of diabetes is higher in patients with more severe COVID-19 (42). Our study cohort had a similar diabetes prevalence (23.6%). Prevalence of A1C >5.7% in our study population was even higher (74.3%) among the 1,532 patients who had A1C measured. Reported rates of diabetes and prediabetes in the general U.S. population (10.5 and 34.5%, respectively) are lower than in studies of patients with COVID-19 (43), pointing to a likely relationship between diabetes and prediabetes and severe COVID-19. The association of diabetes with critical illness and mortality in our study (ORs 1–2) is similar to reports in the literature of a 1.5- to 4.6-fold higher risk of mortality and a 2- to 2.7-fold higher risk of severe disease in patients with COVID-19 and diabetes than in COVID-19 patients without diabetes (10,40,44–52). Our results are consistent with those of previous smaller studies of shorter duration conducted in our health system (17,37). The results from a well-cited multicenter study of patients with diabetes who were hospitalized with COVID-19 in France did not find glycemic control to be an independent predictor of mortality, which contrasts with our study and with published meta-analyses, but 20.3% required MV and 10.6% died as early as 7 days after admission, still suggesting an association of diabetes with poor outcomes (53). Less has been published on prediabetes (A1C 5.7 to <6.5%) or A1C ≥5.7% and outcomes in COVID-19. Smaller studies with 184–453 patients found an association of prediabetes with MV, ICU admission, and mortality (54–56) and an association of A1C ≥5.7%, with higher markers of inflammation and coagulability and lower oxygen saturation (57). The presence of A1C >5.7% in our cohort was found to be associated with MV and ICU admission, but not death. A possible explanation for why elevated A1C was not found to be associated with mortality is that A1C cannot evaluate for glycemic variability and higher glucose levels seen during acute illness, both of which are known to contribute to poor outcomes. Elevated fasting plasma glucose levels have been found to be a better predictor of infection severity and mortality than A1C in COVID-19 patients (55). Comparing studies is difficult because of differences in A1C categories and cutoffs and measured outcomes. Our study differs from previous ones by its inclusion of a larger patient cohort (1,532 patients) who had A1C measured. Postulated reasons for poor outcomes in COVID-19 in patients with elevated A1C and diabetes include the associations of diabetes with older age, other comorbidities, chronic inflammation, higher inflammatory markers, and a prothrombotic state (10,44,48). Higher levels of inflammation-related biomarkers (IL-6, erythrocyte sedimentation rate, CRP, serum ferritin, d-dimer, and fibrinogen) found in patients with COVID-19 and diabetes (58,59) may predispose to an inflammatory cytokine storm, impaired T-cell responses, lung injury, and possibly increased infectivity related to abnormal ACE2 expression and increased furin, all of which are potential underlying mechanisms of more severe COVID-19 in diabetes (60,61). Our findings provide evidence to support greater diabetes screening and use of A1C to detect and allow earlier treatment of prediabetes and diabetes during the COVID-19 pandemic to prevent poor outcomes. BMI and Outcomes From COVID-19 Studies of COVID-19 have described a prevalence of obesity in patients that ranges from 16.3% in Italy to 57.6% in Detroit (8,17,37–39,62–64). Higher obesity prevalence rates have been found in U.S. studies, especially in Detroit (17,37), as reported in our study, with 50.3% of patients in the obese range. Higher obesity prevalence in hospitalized COVID-19 patients than in the general U.S. and Michigan populations (42.4 and 36% in 2017–2018, respectively) (65) suggests an association of obesity with more severe disease. On the other hand, the prevalence of being merely overweight in hospitalized COVID-19 patients (21–37% in various studies; 27.5% in our study cohort) (8,53,66,67) has not been found to be much higher than the general population (31.1% in the United States in 2017–2018 [68] and 34.9% in Michigan in 2014 [69]). Our results demonstrated that obesity and elevated BMI starting even at ≥25.0 kg/m 2(overweight levels) were significantly associated with the need for MV and ICU admission, but not with death or VTE. Most published reports support the association of obesity with critical illness (MV, ICU admission, and acute respiratory distress syndrome) (8,39,40,53,67,70–74), but the association of being overweight with critical illness is less consistent, with only some studies finding a relationship of being overweight with MV (53,66), but not others (8,67,75). The relationship of higher BMI and death in COVID-19 patients is also not clear. Most studies, including a meta-analysis of 33 studies on obesity and COVID-19, the CORONADO (Coronavirus SARS-CoV-2 and Diabetes Outcomes) study of patients with diabetes and COVID-19, a recent Chinese study, and two large studies from New York, found an association of obesity with higher risk of death (8,53,66,67,70); however, several U.S. studies did not find obesity to be associated with mortality from COVID-19 (15,37,39,40). Being overweight was not found to be associated with higher risk of death in the few studies evaluating this weight category (8,66,67). Our results add to this variable literature, supporting the findings that obesity and elevated BMI starting even at the overweight category are associated with critical illness but not death. More research on the effects of weight and BMI on COVID-19 mortality needs to be done. Molecular mechanisms linking obesity with poor prognosis from COVID-19 include 1) alteration in adipose tissue resulting in a proinflammatory state with increased secretion of cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-α, IL-1β, IL-6, and interferon-γ), contributing to risk of cytokine storm; 2) upregulation of ACE2, the functional receptor for SARS-CoV-2, and dipeptidyl peptidase 4, another potential SARS-CoV-2 receptor, facilitating virus entry and a severe inflammatory and immune response; and 3) expression of furin, a priming protease for SARS-CoV-2 in vascular endothelial cells, which may contribute to inflammation, damage to vascular endothelium, and coagulopathy (76). Also, obesity negatively affects lung function through mechanical/restrictive means and excess inflammation (76). Inflammatory Markers and Outcomes From COVID-19 Many studies, including several meta-analyses, have shown a relationship between higher levels of proinflammatory markers such as CRP, IL-6, erythrocyte sedimentation rate, procalcitonin, ferritin, and d-dimer and more severe disease and mortality from COVID-19 (8,39,44,77–83). However, not all studies have shown a strong correlation between inflammatory markers and clinical outcomes in COVID-19 (84,85). Our results added to the literature supporting the inflammatory state as a major mechanism of severe COVID-19. Limitations Our study has several limitations, including its retrospective design. The study population included only patients hospitalized for COVID-19 in one health care system. This cohort may have had additional risk factors for admission that were not measured in this study, such as socioeconomic status or work and home situations, all of which may contribute to severity of illness. Because patients admitted to the hospital tend to be sicker and to have comorbidities, the cohort may not be representative of the general population. Because data collected on patient clinical variables was based on International Classification of Diseases, 10th revision, diagnosis codes, some diagnoses may have been missed if the diagnosis or code was not entered for a patient. Data on BMI and A1C were not present for all patients, although we still had >5,000 patients with BMI recorded and >1,500 with A1C results. We used diagnosis of diabetes instead of elevated A1C, as this method captured more patients with diabetes. Analysis by A1C should be further investigated in large observational studies because of the potential for gathering incomplete data and potential skewing of results, since A1C tends to be measured only in those predicted to have high levels. Conclusion This large retrospective study identified older age, male sex, Black race, diabetes, elevated BMI, and elevated inflammatory markers as potential risk factors for severe COVID-19. Our findings emphasize the need to 1) better identify patients with prediabetes and diabetes through screening with A1C, 2) check for elevated BMI in all patients, and 3) more aggressively address and treat these conditions to reduce risk. The urgency to identify and address diabetes and elevated BMI during and after the pandemic is evident from data indicating that 42% of adults gained almost 30 lb, and almost half delayed or canceled health care services during the pandemic (86). Recently published recommendations for the management of diabetes and obesity during COVID-19 can help providers mitigate risk (87,88). Furthermore, our results indicate a need to work to reduce disparities in health care for Black patients. Increasing the use of telemedicine may help to improve health care equity and reduce barriers. Vaccinations are also now widely available and should be encouraged for those at risk for severe COVID-19, given current lack of definitive treatment for this new infectious disease. Article Information Acknowledgments The authors thank Karla Passalacqua, PhD, of Henry Ford Hospital for her editorial assistance with the manuscript. Duality of Interest No potential conflicts of interest relevant to this article were reported. Author Contributions J.H. researched the data, contributed to writing the manuscript, and reviewed the manuscript. E.T. contributed to the statistical analysis of the data and reviewed and edited the manuscript. S.W.L. researched the data, wrote the manuscript, and reviewed and edited the manuscript. S.W.L. is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. This article is part of a special article collection available athttps://diabetesjournals.org/journals/collection/41/Clinical-Diabetes-COVID-19-and-Diabetes-Patient. References 1. Johns Hopkins University and Medicine . Coronavirus resource center . Available from https://coronavirus.jhu.edu . Accessed 11 June 2021 2. Cutler DM , Summers LH . The COVID-19 pandemic and the $16 trillion virus . JAMA 2020 ; 324 : 1495 – 1496 Google Scholar Crossref Search ADS PubMed 3. Centers for Disease Control and Prevention . COVID-19 pandemic planning scenarios . Available from https://www.cdc.gov/coronavirus/2019-ncov/hcp/planning-scenarios.html . Accessed 22 October 2020 4. Zheng Z , Peng F , Xu B , et al . Risk factors of critical & mortal COVID-19 cases: a systematic literature review and meta-analysis . 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Difference between revisions of "Mifflin County, Pennsylvania Genealogy" • FamilySearch United States Genealogy Pennsylvania, United States Genealogy Mifflin County Difference between revisions of "Mifflin County, Pennsylvania Genealogy" From FamilySearch Wiki Jump to navigation Jump to search Browse history interactively Visual Wikitext Revision as of 12:03, 19 September 2012 ( view source ) Murphynw ( talk \| contribs ) (access) ← Older edit Revision as of 12:04, 19 September 2012 ( view source ) Murphynw ( talk \| contribs ) (access) Newer edit → Line 205: Line 205: ==== Military ==== ==== Military ==== − ===== Civil War ===== + ===== Civil War ===== − :''Mifflin Countians in the Civil War.'' Surname index at [http://www.pagenweb.org/~mifflin/mchs-cw.html PAGenWeb] - free. + : Frysinger, George R. and Eleanor M. Aurand. ''Mifflin Countians Who Served in the Civil War : Published in the Lewistown Gazette 1905 .'' Lewistown, Pa.: Mifflin County Historical Society, 1996. Surname index at [http://www.pagenweb.org/~mifflin/mchs-cw.html PAGenWeb] - free ; {{FHL\|785154\|item\|disp=FHL Book 974.846 M2f}} . ==== Naturalization and Citizenship ==== ==== Naturalization and Citizenship ==== Revision as of 12:04, 19 September 2012 United States Genealogy Pennsylvania, United States Genealogy Mifflin County Mifflin County , Pennsylvania Map Location in the state of Pennsylvania, United States Genealogy Location of Pennsylvania in the U.S. Facts Founded September 19, 1789 County Seat Lewistown Courthouse Address Mifflin County Courthouse 20 North Wayne St. Lewistown, PA 17044 Mifflin County Website Adopt-a-wiki page This page adopted by: PA Roots and its members who welcome you to contribute. Adopt a page today Contents 1 Historical Facts 1.1 Boundary Changes 2 Resources 2.1 Bible Records 2.2 Cemeteries 2.3 Census 2.4 Church Records 2.4.1 County-wide Database – Multi-denominational 2.5 Court Records 2.5.1 Court of Common Pleas 2.5.2 Clerk of the Court 2.5.3 Prothonotary 2.5.4 Orphans' Court 2.6 Emigration and Immigration 2.7 Ethnic Groups 2.7.1 Germans 2.8 Gazetteers 2.9 Genealogy 2.10 History 2.10.1 Published Histories 2.11 Land and Property 2.12 Maps 2.13 Migration 2.14 Military 2.14.1 Civil War 2.15 Naturalization and Citizenship 2.16 Newspapers 2.16.1 Obituaries 2.17 Occupations 2.17.1 Indentured Servants 2.18 Periodicals 2.19 Poorhouse, Almshouse 2.20 Probate Records 2.21 Repositories 2.21.1 Archives 2.21.2 Courthouse 2.21.3 Family History Centers 2.21.4 Libraries 2.21.5 Museums 2.21.6 Societies 2.22 Taxation 2.23 Vital Records 2.23.1 Birth 2.23.2 Marriage 2.23.3 Divorce 2.23.4 Death 3 Web Sites 4 Places 4.1 Populated Places 5 References Historical Facts [ edit \| edit source ] Beginning dates for major county records Birth Marriage Death Deeds Probate 1893 1885 1893 1789 1789 Parent Counties : Formed from Cumberland and Northumberland Counties 19 September 1789. [1] County Seat: Lewistown Neighboring Counties : Mifflin County, Pennsylvania Genealogy residents may also have records in [2] Centre (north) • Huntingdon (west) • Union (northeast) • Juniata (south) Snyder (east) Named for Thomas Mifflin , the first Governor of the state of Pennsylvania. Boundary Changes [ edit \| edit source ] 13 February 1800: Centre County was set off. [1] 2 March 1831: Juniata County was set off. [1] Resources [ edit \| edit source ] Bible Records [ edit \| edit source ] Cemeteries [ edit \| edit source ] Cemetery records often reveal birth, marriage, death, relationship, military, and religious information. Online Grave Transcripts Published Grave Transcripts County Cemetery Directories Findagrave.com Family History Library Findagrave.com Interment.net WorldCat Names in Stone USGenWeb-transcripts USGenWeb-tombstone photos Billion Graves Pennsylvania Genealogy Epodunk Pennsylvania Gravestones Billion Graves Names in Stone Access Genealogy Ancestry($) See Pennsylvania Cemeteries for more information. The Cemeteries of Mifflin County. 2 vols. Mifflin County Historical Society, 1977. Surname index and list of cemeteries at PAGenWeb - free; FHL Book 974.846 V22m v. 1-v. 2 . Census [ edit \| edit source ] For tips on accessing Mifflin County, Pennsylvania Genealogy Federal (or United States) census records online, see: Pennsylvania Census . There are no county or state census records available for Pennsylvania. County and city tax records can be used as a substitute when census records are not available. Church Records [ edit \| edit source ] Church records and the information they provide vary significantly depending on the denomination and the record keeper. They may contain information about members of the congregation, such as age, date of baptism, christening, or birth; marriage information and maiden names; and death date. In Pennsylvania, church records are often used as a substitute for birth, marriage, and death information. For general information about Pennsylvania denominations, view the Pennsylvania Church Records wiki page. Finding Church Records at Other Repositories Additional church records can sometimes be found using search phrases such as Mifflin County, Pennsylvania Genealogy Church Records in online catalogs like: Historical Society of Pennsylvania WorldCat (For instructions see WorldCat Online Catalog ). FamilySearch Catalog (For instructions see FamilySearch Catalog Places Search ). County-wide Database – Multi-denominational [ edit \| edit source ] 1708-1985 - Pennsylvania, Church and Town Records, 1708-1985 at Historical Society of Pennsylvania – $, free to members of the society; Also available at Ancestry.com –$; 7,542,774 entries. This database is incomplete for all counties. Contains the church records of: Burnham: United Methodist Burnham Charge; United Methodist Church Lewistown: First United Methodist Church; Grace United Methodist Church; United Methodist Church Reedsville: United Methodist Church Yeagertown: United Methodist Church Court Records [ edit \| edit source ] Mifflin County, Pennsylvania Genealogy court records are housed at the Mifflin County, Pennsylvania Genealogy Courthouse . For many counties copies of court records may be found at the Pennsylvania State Archives and in the FamilySearch collection. Note that within these collections some films may contain the same records, but have different titles. Other titles are not duplicates. See Finding Court Records at other repositories within this section for links to the online catalogs for these two collections. Films at the Pennsylvania State Archives are not available for inter-library loan. If court records are available FamilySearch films may be available at a local FamilySearch Center . Call ahead for availability. Court of Common Pleas [ edit \| edit source ] The Courts of Common Pleas are the trial courts of Pennsylvania. Major civil and criminal cases are heard in these courts. Judges also decide cases involving adoption, divorce, child custody, abuse, juvenile delinquency, estates, guardianships, charitable organizations and many other matters. The Common Pleas courts are organized into 60 judicial districts. Mifflin County, Pennsylvania Genealogy has its own judicial district. Judges of the Common Pleas courts are elected to 10-year terms. A president judge and a court administrator serve in each judicial district. [3] Clerk of the Court [ edit \| edit source ] The Clerk of Courts prepares and maintains the records for the Criminal Division of the Court of Common Pleas. The Clerk signs and affixes the Seal of the Courts to all writs and processes, administers oaths and affirmations, and assumes custody of the seal and records of the Courts. The Clerk certifies and distributes orders of the Court. The Clerk also certifies and prepares bills of costs for the defendants and utilizes the computerized financial management system to disburse fines, costs and restitution. [4] For the Mifflin County, Pennsylvania Genealogy Clerk of Courts address see the Courthouse section on this page. Prothonotary [ edit \| edit source ] The office of the Prothonotary is the custodian of all civil matters in the county. This includes naturalization, immigration, equity actions, judgements, federal and local tax liens, city liens, family court, arbitrations, license suspension appeals, appeals to higher court, commercial code filings, applications for passports and divorce proceedings. See the Courthouse section on this page for the Mifflin County, Pennsylvania Genealogy Prothonotary office information. The Prothonotary and the Clerks offices are combined. The Prothonotary is listed with the Elected Officials Offices. Orphans' Court [ edit \| edit source ] Emigration and Immigration [ edit \| edit source ] For information about emigration into Pennsylvania, see the Pennsylvania Emigration and Immigration page. For residents who moved away, see: McClenahen, Dan. Former Residents of Mifflin County 1832-1883, 1894-1896, 2 vols. Reedsville, Pa.: D. McClenahen, 1986-1993. Surname index at PAGenWeb - free; FHL Books 974.846 W2m v. 1-v. 2 . Ethnic Groups [ edit \| edit source ] Germans [ edit \| edit source ] Gazetteers [ edit \| edit source ] Genealogy [ edit \| edit source ] Bell, Raymond Martin. Families and Records Before 1800. 1987, 1991. Includes supplement. Surname and place index at PAGenWeb - free. History [ edit \| edit source ] Published Histories [ edit \| edit source ] Sherman Day's "Mifflin County, Historical Collections of the State of Pennsylvania, 1843 , courtesy USGenWeb Archives Land and Property [ edit \| edit source ] Land records in Mifflin County began in 1789. These records are filed with the Recorder of Deeds office in Lewistown, Pennsylvania. Land and property records can place an ancestor in a particular location, provide economic information, and reveal family relationships. Land records include: deeds, abstracts, indexes, mortgages, leases, grants, sheriff sales, land patents, and maps. Property records include liens as well as livestock brands and estray records. The following are examples of available resources: Land Records on Microfilm 1789-1953 Deed Indexes. FHL film 1026092 (first of 10 films) Additional Resources Note that the "Maps" section below also includes maps related to land ownership. See Pennsylvania Land and Property for more information about using land records, especially about original land warrants, surveys, and patents filed at the state land office. Additional resources can sometimes be found using search phrases such as Mifflin County Pennsylvania Land in online catalogs such as: Historical Society of Pennsylvania WorldCat (For instructions see WorldCat Online Catalog .) Family History Library Catalog (For instructions see FHL Catalog Place-name Search .) Maps [ edit \| edit source ] Ancestor Tracks has posted free downloadable images from the Atlas of Perry, Juniata, and Mifflin Counties by and under the Direction of Beach Nichols, published by Pomery, Whitman & Co., 1877 . This atlas located in the Library of Congress shows major landowners and geographic sites at the date of publishing. While the physical maps are in the public domain, the images we have taken of the maps belong to us and are not to be used commercially. We hereby give permission to use them strictly for personal use; please attribute to Ancestor Tracks. Migration [ edit \| edit source ] Military [ edit \| edit source ] Civil War [ edit \| edit source ] Frysinger, George R. and Eleanor M. Aurand. Mifflin Countians Who Served in the Civil War: Published in the Lewistown Gazette 1905. Lewistown, Pa.: Mifflin County Historical Society, 1996. Surname index at PAGenWeb - free; FHL Book 974.846 M2f . Naturalization and Citizenship [ edit \| edit source ] Naturalization records can contain information about immigration and nativity. Prior to 1906, it is rare to find the town of origin in naturalization records. See Pennsylvania Naturalization for more information about the types of records and availability. Naturalizations granted at the county level were kept by the office of the Prothonotary . Naturalizations could also be granted on the Federal Court level. 1803-1906 - McClenahen, Dan. Naturalization Papers of Mifflin County, 1803-1906. Lewistown, Pa.: Mifflin County Historical Society, 1984. Surname index at PAGenWeb - free; FHL Book 974.846 P4m . Naturalization records available for Mifflin County, Pennsylvania include the following: Online Naturalization Indexes and Records 1901-1930 – Naturalization Petitions of the U.S. Circuit and District Courts for the Middle District of Pennsylvania, 1906-1930; and 1901-1906 on Fold3.com - ($) , indexed, National Archives publication M1622, Middle District court was created in 1901, for earlier dates try the Eastern and Western District court records 1901-1930 - Middle District, US Circuit and District Courts: Naturalization petitions, 1906-1930 located in U.S. Naturalization Records - Original Documents, 1795-1972 (World Archives Project) database on Ancestry.com - ($) , indexed, National Archives publication M1622, Middle District court was created in 1901, for earlier dates try the Eastern and Western District court records Newspapers [ edit \| edit source ] Mifflin County, Pennsylvania Genealogy newspapers may contain genealogical value including obituaries, births, marriages, deaths, anniversaries, family gatherings, family travel, achievements, business notices, engagement information, and probate court proceedings. To access newspapers, contact public libraries , historical/genealogical societies , college or university libraries , or state archives in the area where the newspaper was published. For information on state-wide newspapers see Pennsylvania Newspapers Newspapers of Mifflin County Pennsylvania Newspapers Chronicling America US Newspaper Directory Online Newspapers To learn if there are newspapers online for a specific town or city in Pennsylvania, see news.google.com/newspapers and search for the town or the name of a newspaper. Online Newspaper Abstracts PA-Roots Newspaper Articles for Mifflin County, Pennsylvania. Newspaper Abstracts alphabetical. Also Church Newsletter Abstracts. Newspaper Excerpts and Abstracts McClenahen, Dan, People in the News in Mifflin County (S.I.:D. McDlenahen, 1986-1995) At various libraries (WorldCat) ; FHL book 974.846 D2m Obituaries [ edit \| edit source ] Obituaries are generally found in local newspapers where the person died. However, sometimes an obituary is found in the location from which he or she originated. To find an obituary, see the information under the Newspaper heading Sometimes the fastest way of finding obituaries is to call or email the local public library in the area where the person died. If the library does not have newspapers, a librarian often will know where they are kept. If a death date is known, and the newspapers are at the library, someone on staff will usually make a search for a small fee, or will indicate someone who will do the search. Online Obituary Abstracts PA-Roots Obituaries for Mifflin County, Pennsylvania. Mifflin County Obituaries at USGenWeb Archives Obituary Excerpts and Abstracts 1822-1896 - McClenahen, Dan, Obituaries of Mifflin County 1822-1896, 2 vols. Lewistown, Pa.: Mifflin County Historical Society, 1982-1995. Surname index at PAGenWeb - free; At various libraries (WorldCat) ; FHL book 974.846 V4m Occupations [ edit \| edit source ] Indentured Servants [ edit \| edit source ] Periodicals [ edit \| edit source ] Poorhouse, Almshouse [ edit \| edit source ] Probate Records [ edit \| edit source ] Probate matters in {{{1}}} County are handled by the Orphans' Court and start when the county was created. In addition to wills and administrations, the Orphans' Court also handles: audits of accounts of executors, administrators, trustees, and guardians; distribution of estates; appointments of guardians; adoptions; appeals from the Register of Wills; inheritance tax appeals, and various petitions and motions. Online Probate Records Original probate records for some Pennsylvania counties are available free online as digital images at FamilySearch.org . The dates vary significantly for each county and not all counties are listed. Some counties may only have probate indexes. This Pennsylvania collection of images may be browsed through the links listed below: Complete Collection: 1683-1994 – Pennsylvania, Probate Records, 1683-1994 at FamilySearch.org Mifflin County Only: 1789–1899 – Mifflin County Probate Records Fisher, Charles A. Wills and Administrations of Northumberland County, Pennsylvania. Including Wills and Administrations of Union, Mifflin and Indiana Counties. 1950. Digital version at FamilySearch Digital Library - free. Abstracts and index. 1789-1900 - McClenahen, Dan. Wills of Mifflin County, 1789-1900, 2 vols. 1983-1985. Surname index at PAGenWeb - free; FHL Books 974.846 P2m v. 1-v. 2 . Original Probate Records on Microfilm 1789-1899 Register's Docket FHL film 1026537 Template:PA probate FHLC and WorldCat Repositories [ edit \| edit source ] Archives [ edit \| edit source ] The Pennsylvania Archives collection contains county archive records that can be searched onsite. Currently the Archives' staff cannot provide research or make copies of these records. Their collections include Almshouse Registers; Tax records; Birth, Death and Marriage Indexes and Records; Midwife records; African American records; Wills; Deeds; Naturalizations; Coroner's inquests; and Orphan's Court dockets. A list of the Archive's county holdings are on Microfilm or Manuscript form . Courthouse [ edit \| edit source ] Mifflin County Courthouse 20 North Wayne St. Lewistown, PA 17044 Clerk of Courts/Prothonotary Phone: 717–248-8146 E–mail: prothonotary@co.mifflin.pa.us Register, Recorder and Clerk of the Orphans' Court Phone: 717–242-1449 Family History Centers [ edit \| edit source ] FamilySearch Centers Libraries [ edit \| edit source ] Museums [ edit \| edit source ] Societies [ edit \| edit source ] Rich in history, Mifflin County, Pennsylvania Genealogy contains a number of societies dealing with the history of the area. The Mifflin County Historical Society 1 West Market Street, Suite 1 Lewistown, PA 17044 Phone: (717) 242-1022 Hours: Tuesdays & Wednesdays 10:00 a.m. to 4:00 p.m. March 1st through Mid November 17. Seasonal hours The historical society has a research room and will do research in their facility or at the courthouse for a fee. Mifflin County Mennonite Historical Society Phone: 717–935-5574 The Heritage Center is open mid-April through December, Wednesdays and Saturdays 9:00 am to 12:00 noon. January to mid-April it is open only on Saturday. Persons may use the center at other times by appointment. Taxation [ edit \| edit source ] 1767-1789, 1798 - The People of Mifflin County, Pennsylvania, 1755-1798: Tax Lists 1767-1789; Federal Census 1790; Federal Direct Tax 1798 at FamilySearch Digital Library - free; surname index at PAGenWeb - free. 1798 - Pennsylvania, U.S. Direct Tax Lists, 1798 at Ancestry ($) . Vital Records [ edit \| edit source ] Vital records are handled by the County Orphans' Court. Between the years 1852-1855 Pennsylvania made a failed attempt to record birth, marriage and death events at the county level. While the records for that time period are available, there were few events recorded. County marriage records were kept in earnest in 1885. Births and deaths, at the county level, were begun in 1893 and kept through 1905. Abstracts and copies of vital records are available for some counties, but most are incomplete. For the most complete set of records, always contact the County Orphans' Court. Birth [ edit \| edit source ] 1726-1950 Pennsylvania Births and Christenings, 1709-1950 - free. This index is an electronic index for the years 1726 to 1930. It is not necessarily intended to index any specific set of records. This index is not complete for any particular place or region. This collection may include information previously published in the International Genealogical Index or Vital Records Index collections. 1852-1854 – Pennsylvania Births Ancestry.com – ($) Index with images. Early births 1893–1905 are located at the County Orphans' Court. See the heading Court Records on this page for contact information. Indexes for Pennsylvania birth records are available through the Department of of Health for 1906 and 1907. Once an individual is located in the index a non certified Birth certificate can be obtained by writing and sending $3.00 to: Division of Vital Records ATTN: Public Records P.O. Box 1528 New Castle, PA 16103 Marriage [ edit \| edit source ] Pennsylvania marriages are located at the county level. Contact the Mifflin County, Pennsylvania Genealogy clerk's office for these records. 1700-1821 - Pennsylvania Marriage Records Ancestry.com – ($) This database is incomplete for all counties. 1725-1976 - Pennsylvania, Marriages, 1709-1940 - free index. Not complete for all years. This index is an electronic database of information. The entries are primarily from the International Genealogical Index ( IGI ) along with some entries derived from compiled and original records such as: Family Records, Church Records, Civil Registration. It may also include indexes generated by the internet indexing project sponsored by the LDS Church. Pre-1810 – Pennsylvania Marriages Ancestry.com – ($) This database is incomplete for all counties. Includes 35,000 marriage records from vol. VIII of of the second series of the Pennsylvania Archives. 1822-1885 - McClenahen, Dan. Marriages of Mifflin County, 1822-1885. Lewistown, Pa.: Mifflin County Historical Society, 1981. Surname index at PAGenWeb - free. 1852-1854 - Pennsylvania Marriages Ancestry.com – ($) Index with images. Divorce [ edit \| edit source ] Divorce records are available through the office of the Prothonotary. The office of the Prothonotary is located in the courthouse building. Death [ edit \| edit source ] 1852-1854 – Pennsylvania Deaths Ancestry.com – ($) Index with images. Early deaths 1893–1905 are located at the County Orphans' Court. See the heading Court Records on this page for contact information. Indexes for Pennsylvania death records are available through the Department of Health for 1906 through 1962. Once an individual is located in the index a non certified death certificate can be obtained obtained by writing and sending $3.00 to: Division of Vital Records ATTN: Public Records P.O. Box 1528 New Castle, PA 16103 Finding Vital Records at Other Repositories Additional vital records can sometimes be found using search phrases such as Mifflin County, Pennsylvania Genealogy Vital Records in online catalogs like: Historical Society of Pennsylvania WorldCat (For instructions see WorldCat Online Catalog ). FamilySearch Catalog (For instructions see FamilySearch Catalog Places Search ). Web Sites [ edit \| edit source ] USGenWeb project. May have maps, name indexes, history or other information for this county. Select the state, then the county. Family History Library Catalog Places [ edit \| edit source ] Populated Places [ edit \| edit source ] References [ edit \| edit source ] ↑ 1.0 1.1 1.2 Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), [ FHL book 973 D27e 2002]. Cite error: Invalid <ref> tag; name "HBG" defined multiple times with different content Cite error: Invalid <ref> tag; name "HBG" defined multiple times with different content ↑ Wikipedia contributors, "Adams County, Pennsylvania" in Wikipedia: the Free Encyclopedia at http://en.wikipedia.org/wiki/Adams_County,_Pennsylvania (accessed 17 July 2012). ↑ The Unified Judicial System of Pennsylvania http://www.pacourts.us/T/CommonPleas/ accessed 10 July 2012. ↑ Unified Judicial System of Pennsylvania, Clerk of Courts in http://www.pacourts.us/T/CommonPleas/ClerksOfCourts.htm (accessed 25 Aug 2012) v • d • e Links to Pennsylvania -related articles Topics Search Strategies · Record Finder · Introduction · American Indians · Archives and Libraries · Bible Records · Biography · Cemeteries · Census · Church Records · Court Records · Directories · Emigration and Immigration · Ethnic, Political, or Religious Groups · Gazetteers · Genealogy · Historical Geography · History · Land and Property · Law and Legislation · Maps · Military Records · Native Races · Naturalization and Citizenship · Newspapers · Occupations · Online Records · Periodicals · Probate Records · Societies · Taxation · Vital Records · For Further Reading Counties Adams · Allegheny · Armstrong · Beaver · Bedford · Berks · Blair · Bradford · Bucks · Butler · Cambria · Cameron · Carbon · Centre · Chester · Clarion · Clearfield · Clinton · Columbia · Crawford · Cumberland · Dauphin · Delaware · Elk · Erie · Fayette · Forest · Franklin · Fulton · Greene · Huntingdon · Indiana · Jefferson · Juniata · Lackawanna · Lancaster · Lawrence · Lebanon · Lehigh · Luzerne · Lycoming · McKean · Mercer · Mifflin · Monroe · Montgomery · Montour · Northampton · Northumberland · Perry · Philadelphia · Pike · Potter · Schuylkill · Snyder · Somerset · Sullivan · Susquehanna · Tioga · Union · Venango · Warren · Washington · Wayne · Westmoreland · Wyoming · York Extinct Co. 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Microscopic Electrical Characterization of Inorganic Semiconductor-Based Solar Cell Materials and Devices Using AFM-Based Techniques Download Citation \| Microscopic Electrical Characterization of Inorganic Semiconductor-Based Solar Cell Materials and Devices Using AFM-Based Techniques \| Atomic force microscopy (AFM)-based microscopic electrical techniques provide new insights into the characterization of electronic devices, which... \| Find, read and cite all the research you need on ResearchGate Microscopic Electrical Characterization of Inorganic Semiconductor-Based Solar Cell Materials and Devices Using AFM-Based Techniques November 2011 NanoScience and Technology 116 DOI: 10.1007/978-3-642-10497-8_24 Authors: Abstract Atomic force microscopy (AFM)-based microscopic electrical techniques provide new insights into the characterization of electronic devices, which is useful for understanding device physics and improving device performance. This chapter introduces the fundamentals of some major AFM-based electrical techniques, including scanning Kelvin probe force microscopy (SKPFM), scanning capacitance microscopy (SCM), and conductive AFM (C-AFM), and reviews some recent characterization studies on inorganic semiconductor solar cell materials and devices. A potential measurement on junctions of solar cells presents SKPFM as a powerful tool for two-dimensional junction location identification by direct electrical measurement. The potential measurement further reveals electrical field distributions under bias voltages that relate to the defects on the junctions. Grain boundaries (GBs) of polycrystalline thin-film materials are characterized by measuring carrier depletion or band bending using SCM and SKPFM, which provide the measurements of individual GBs and are thus able to correlate the GB electrical properties directly to the GB structure. Structural and electrical properties of a-Si:H and nc-Si:H thin films and devices are also characterized through a prototype structure of a-Si:H and nc-Si:H mixed phase. Localized electrical properties on the nc-Si:H phase, and phosphorus and boron doping effects on the film structure are reviewed. Semiconductor Materials and Modelling for Solar Cells Apr 2021 Zahra Pezeshki Abdelhalim abdelnaby Zekry The book presents a comprehensive survey about advanced solar cell technologies. Focus is placed on semiconductor materials, solar cell efficiency, improvements in surface recombination velocity, charge density, high ultraviolet (UV) sensitivity, modeling of solar cells etc. The book references 281 original resources with their direct web links for in-depth reading. Characterization of grain boundaries in multicrystalline silicon with high lateral resolution using conductive AFM, Full-text available Aug 2012 Maximilian Rumler Mathias Rommel Jürgen Erlekampf Lothar Frey In this work, the electrical characteristics of grain boundaries (GBs) in multicrystalline silicon with and without iron contamination are analyzed by fixed voltage current maps and local I/V curves using conductive AFM (cAFM). I/V characteristics reveal the formation of a Schottky contact between the AFM tip and the sample surface. The impact of both, the polarity of the applied voltage and the illumination by the AFM laser on the behavior of GBs was analyzed systematically. Depending on the polarity of the applied voltage and the iron content of the sample, grain boundaries alter significantly the recorded current flow compared to the surrounding material. The results also show a clear influence of the AFM laser light on the electrical behavior of the grain boundaries. Conductive AFM measurements are furthermore compared to data obtained by electron beam induced current (EBIC), indicating that cAFM provides complimentary information. Micro-Raman Studies of Mixed-phase Hydrogenated Silicon Solar Cells Jan 2003 Jessica M. Owens Daxing Han Baojie Yan Subhendu Guha The open-circuit voltage (V oc) of mixed-phase hydrogenated silicon solar cells has been found to increase after light soaking. In this study, we use micro-Raman to investigate the heterogeneous structure of solar cells in the amorphous-to-nanocrystalline transition region. For a cell with V oc = 0.981 V, Raman spectra show a typical broad Gaussian lineshape around 480 cm -1 , a signature of typical amorphous material. A cell with V oc = 0.674 V displays a sharp Lorentzian peak around 516 cm -1 , indicative of nanocrystallinity. A cell with V oc = 0.767 V was systematically scanned for 20 different positions in 500 µm increments. Most spectra show a typical Gaussian lineshape around 480 cm -1 , several spectra reveal a hint of a nanocrystalline shoulder around 512 cm -1 , and one spectrum exhibits a distinct nanocrystalline peak. We conclude that the nanocrystallite distribution in the mixed-phase material is very non-uniform even within a mm dot. This result provides direct evidence supporting a recently proposed two-diode equivalent-circuit model to explain the light-induced effect. Theoretical model of interface trap density using the spread of the differential capacitance characteristics in scanning capacitance microscopy measurements Feb 2006 K.M. Wong Kin Mun Wong In this letter, we propose a theoretical model for the calculation of interface trap density (Dit) in a metal-oxide-semiconductor structure using data from scanning capacitance microscopy (SCM) measurements. The model is based on the correlation of Dit with the change in the full width at half maximum of the SCM differential capacitance (dC/dV) characteristics. The good agreement between the calculated Dit values from the SCM theoretical model and the experimental midgap Dit values obtained from conductance measurements shows the validity of the proposed model. The model opens up possibilities for obtaining the spatial distribution (with nanometers resolution) of interfacial traps on a device using SCM measurements. Local built-in potential on grain boundary of Cu(In, Ga)Se2 thin film Apr 2004 APPL PHYS LETT J. AbuShama C.-S. Jiang R. Noufi Mowafak Al-Jassim We report on a direct measurement of two-dimensional potential distribution on the surface of photovoltaic Cu(In,Ga)Se2 thin films using a nanoscale electrical characterization of scanning Kelvin probe microscopy. The potential measurement reveals a higher surface potential or a smaller work function on grain boundaries of the film than on the grain surfaces. This demonstrates the existence of a local built-in potential on grain boundaries, and the grain boundary is positively charged. The local built-in potential on the grain boundary is expected to increase the minority-carrier collection area from one to three dimensional. In addition, a work function decrease induced by Na on the film surface was observed. The Atomic Force Microscope Mar 1986 Gerd Binnig Ch. Gerber The scanning tunneling microscope is proposed as a method to measure forces as small as 10-18 N. As one application for this concept, we introduce a new type of microscope capable of investigating surfaces of insulators on an atomic scale. The atomic force microscope is a combination of the principles of the scanning tunneling microscope and the stylus profilometer. It incorporates a probe that does not damage the surface. Our preliminary results in air demonstrate a lateral resolution of 30 ÅA and a vertical resolution less than 1 Å. Conduction band offsets in ordered‐GaInP/GaAs heterostructures studied by ballistic‐electron‐emission microscopy Article Full-text available Nov 1996 APPL PHYS LETT J. J. O'Shea C. M. Reaves Steven Denbaars V. Narayanamurti Ordered‐GaInP/GaAs heterostructures have been studied using ballistic‐electron‐emission microscopy (BEEM). The GaInP/GaAs conduction band offset was found to decrease with increasing order. Samples were grown simultaneously on different misoriented substrates to vary the degree of order in the GaInP. Concurrent scanning tunneling microscopy and BEEM images show ridge structures in the topography and contrast in the BEEM current that may correspond to ordered domains in the GaInP. Room temperature conduction band offsets of 137 and 86 meV were measured using BEEM spectroscopy for GaInP with 2 K band gaps of 1.97 and 1.89 eV, respectively. © 1996 American Institute of Physics. Photovoltaic Materials Book May 1998 Richard H. Bube Jan 2000 Adam J. Schwartz Mukul Kumar Brent L Adams Electron backscatter diffraction (EBSD), when employed as an additional characterization technique to a scanning electron microscope (SEM), enables individual grain orientations, local texture, point-to-point orientation correlations, and phase identification and distributions to be determined routinely on the surfaces of bulk polycrystalline materials. The application has experienced rapid acceptance in metallurgical, materials, and geophysical laboratories within the past decade due to the wide availability of SEMs, the ease of sample preparation from the bulk, the high speed of data acquisition, and the access to complimentary information about the microstructure on a submicron scale. This entirely new second edition describes the complete EBSD technique, from the experimental set-up, representations of textures, and dynamical simulation, to energy-filtered, spherical, and 3-D EBSD, to phase identification, in situ experiments, strain mapping, and grain boundary networks, to the design and modeling of materials microstructures. Numerous application examples including the analysis of deformation microstructure, dynamic deformation and damage, and EBSD studies in the earth sciences provide details of this powerful materials characterization technique. Semiconductors: Other than Group IV Elements and III–V Compounds Book Jan 1992 O. Madelung In the New Series of Landolt-Bomstein the eleven volumes III/17a···i and III/22a,b present data on the properties of semiconductors on more than 6 000 pages with tables and (about 10 000) figures. The aim of the Series "Data in Science and Technology" is to built a bridge between the libraries, where such comprehensive handbooks are situated, and the laboratory. The first volume of this series, published in 1991, contains data on the most important groups of semiconductors, the group IV elements and the III-V compounds. From the wealth of data in the tables and figures of the Landolt-Bornstein volumes III/17a and III/22a,b about 10% were condensed into this first volume. It seemed not appropriate to condense to the same extent all the other material of the remaining nine subvolumes into several further DST volumes. Instead of it all remaining data have been put into the present volume. To do this some severe restrictions became necessary. They are explained in the Introduction, and the reader is asked to read it before using this book. I would further like to focus the attention of the reader on chapter 8, where the bridge between this DST volume and the LB sources is built by cross-references between both books. I do hope that this volume meets the needs of the physical community as a quick reference to basic semiconductor data and an access to the larger data collections on this field of physics. SILICON ( Si ): In 2 Volumes Chapter Nov 1996 Michael E. Levinshtein Sergey L. Rumyantsev CAPACITIVE-PICKUP CIRCUITRY FOR VIDEODISCS. Richard C. Palmer Edgar J. Denlinger Hiro Kawamoto Circuits for capacitive pickup from a VideoDisc were designed using the concept of transmission lines printed on circuit boards. Circuits of the printed-board type have achieved an accuracy required in microwave design and a reproducibility needed in mass production. A theoretical analysis has revealed that the pickup sensitivity increases with operating frequency, which gives an advantage to the design with the microwave transmission-line circuit over an earlier design using a 250-MHz lumped-element circuit. The pickup circuit operating at 915 MHz has generated an output of 10 mv peak-to-peak from a signal capacitance as small as 10 minus 4 pF, and a ratio of carrier-to-circuit noise as high as 68 db measured in a 30-kHz bandwidth. Electron-Beam-Induced Current Study of Grain Boundaries in Multicrystalline Silicon Article Nov 2004 Jun Chen Takashi Sekiguchi Deren Yang Sadahiro Tsurekawa The effects of grain boundary (GB) character and impurity contamination on the recombination activity of grain boundaries (GBs) in multicrystalline silicon (mc-Si) were systematically studied through an electron-beam-induced current (EBIC) technique. First, clean GBs of various characters were checked at 300 and 100 K. The EBIC contrasts of these GBs were in the same range of 0%-2% at 300 K and 2%-4% at 100 K, suggesting that the recombination activity of clean GBs is weak and the GB character has no significant effect on it. Second, the effect of impurities was studied by comparing the EBIC contrasts of the same type of the GBs in mc-Si with different Fe contamination levels. The recombination activity of GBs became stronger as the contamination level rose. The variation in the recombination activity related to the GB character was also observed in these specimens. The random or high-Sigma GBs showed a stronger EBIC contrast than the low-Sigma GBs. Moreover, we found that the EBIC contrast was not uniform along one Sigma3 GB. In a clean mc-Si, the variation in the EBIC contrast of different parts of the Sigma3 GB was 1% at 300 K, whereas it was more than 10% in the contaminated mc-Si. This indicates that not only the GB character but also the GB plane would affect the recombination activity of GBs. These results suggest that the recombination activity of GBs is principally determined by the gettering ability of GBs. Specifically, that random GBs may have the strongest gettering ability, whereas Sigma3 {111} has the weakest. (C) 2004 American Institute of Physics. Imaging Electron Transport across Grain Boundaries in an Integrated Electron and Atomic Force Microscopy Platform: Application to Polycrystalline Silicon Solar Cells Article Jan 2011 Mater Res Soc Symp Proc Fude Liu Oliver Kunz Manuel J. Romero We have investigated the local electron transport in polycrystalline silicon (pc-Si) thin-films by atomic force microscopy (AFM)-based measurements of the electron-beam-induced current (EBIC). EVA solar cells are produced at UNSW by EVAporation of a-Si and subsequent solid-phase crystallization –a potentially cost-effective approach to the production of pc-Si photovoltaics. A fundamental understanding of the electron transport in these pc-Si thin films is of prime importance to address the factors limiting the efficiency of EVA solar cells. EBIC measurements performed in combination with an AFM integrated inside an electron microscope can resolve the electron transport across individual grain boundaries. AFM-EBIC reveals that most grain boundaries present a high energy barrier to the transport of electrons for both p-type and n-type EVA thin-films. Furthermore, for p-type EVA pc-Si, in contrast with n-type, charged grain boundaries are seen. Recombination at grain boundaries seems to be the dominant factor limiting the efficiency of these pc-Si solar cells. Investigation of Charge Trapping at Grain Boundaries in Polycrystalline and Multicrystalline Silicon Solar Cells Article Jan 2011 Mater Res Soc Symp Proc Jennifer T. Heath Chun-Sheng Jiang Helio Moutinho Mowafak Al-Jassim Scanning capacitance microscopy (SCM) often shows a change in contrast at grain boundaries [1-3]. The origins of this contrast and the efficacy of SCM as a tool to identify band bending at grain boundaries in pc-Si and mc-Si are discussed. Contrast at these grain boundaries could be influenced by different oxide growth rates or by defect states at the oxide interface. In order to determine the influence of such mechanisms on the SCM signal, such effects must be modeled; we show that a simple one-dimensional model agrees well with more detailed models of SCM signal strength and indicates, for example, that very small changes in oxide thickness measurably affect the SCM signal. In our experimental data, the uniformity and quality of the oxide layer are confirmed, and increased contrast consistent with depletion regions is still observed at higher order grain boundaries as identified by electron backscattering diffraction, including 9 and 27a. Scans of the SCM signal as a function of dc probe voltage allow such regions to be more quantitatively investigated. The Effect of Dominant Junction on the Open Circuit Voltage of Amorphous Silicon Alloy Solar Cells. Article Jan 2011 Mater Res Soc Symp Proc Adam Pawlikiewicz Subhendu Guha Structure of {112} Σ3 boundary in silicon and diamond Article May 2001 SCRIPTA MATER Hidetaka Sawada H Ichinose The incoherent grain boundary of silicon and diamond in atomic dimension is investigated using a high voltage super resolution electron microscope (HRTEM). A polycrystalline silicon film is produced by vacuum evaporation at ultra high vacuum. It is sliced, polished and ion milled. Advances in experimental technique for quantitative two-dimensional dopant profiling by scanning capacitance microscopy Article Jan 1999 Vladimir V. Zavyalov J. S. McMurray Clayton C. Williams Several advances have been made toward the achievement of quantitative two-dimensional dopant and carrier profiling. To improve the dielectric and charge properties of the oxide–silicon interface, a method of low temperature heat treatment has been developed which produces an insulating layer with consistent quality and reproducibility. After a standard polishing procedure is applied to cross-sectional samples, the samples are heated to 300 °C for 30 min under ultraviolet illumination. This additional surface treatment dramatically improves dielectric layer uniformity, scanning capacitance microscopy (SCM) signal to noise ratio, and C–V curve flat band offset. Examples of the improvement in the surface quality and comparisons of converted SCM data with secondary ion mass spectrometry (SIMS) data are shown. A SCM tip study has also been performed that indicates significant tip depletion problems can occur. It is shown that doped silicon tips are often depleted by the applied SCM bias voltage causing errors in the SCM measured profile. Worn metal coated and silicided silicon tips also can cause similar problems. When these effects are tested for and eliminated, excellent agreement can be achieved between quantitative SCM profiles and SIMS data over a five-decade range of dopant density using a proper physical model. The impact of the tip size and shape on SCM spatial accuracy is simulated. A flat tip model gives a good agreement with experimental data. It is found that the dc offset used to compensate the C–V curve flat band shift has a consistently opposite sign on p- and n-type substrates. This corresponds to a positive surface on p-type silicon and to a negative surface on n-type silicon. Rectification of the large capacitance probing voltage is considered as a mechanism responsible for the apparent flat band shift of (0.4–1) V measured on the samples after heating under UV irradiat ion. To explain the larger flat band shift of (1–5) V, tip induced charging of water-related traps is proposed and discussed. © 1999 American Institute of Physics. Growth of InxGa1-xAs/GaAs heterostructures using Bi as a surfactant May 2000 Manoj R. Pillai Seong-Soo Kim S. T. Ho Scott A Barnett The effects of a bismuth surfactant layer on the molecular beam epitaxy of GaAs and InxGa1-xAs layers on GaAs (001) were studied. The InxGa1-xAs surface reconstruction changed from arsenic stabilized 2×4 to bismuth stabilized 1×3 for high enough bismuth fluxes and low enough substrate temperatures. Maintaining a bismuth stabilized surface during InxGa1-xAs growth resulted in a larger number of reflection high-energy electron diffraction (RHEED) oscillations. RHEED patterns were also streakier after InxGa1-xAs growth with Bi. Roughness measurements using atomic force microscopy showed reduced root mean square roughness with Bi, e.g., from 3.8 to 2.8 nm, for 4 nm thick In0.3Ga0.7As layers. Simulations of x-ray diffraction results from 10 period In0.5Ga0.5As/GaAs superlattices showed that Bi reduced interface roughness from 1.1 to 0.5 nm and reduced interfacial broadening from 2.8 to 2.1 nm. The latter was attributed to reduced In segregation. InxGa1-xAs/GaAs (x=0.2–0.4) multiple quantum wells grown with Bi exhibited photoluminescence peaks that were more intense than those grown without Bi. © 2000 American Vacuum Society. Progress Toward 20% Efficiency in Cu(In,Ga)Se2 Polycrystalline Thin-Film Solar Cells Article Jul 1999 Miguel A. Contreras Brian Egaas K. Ramanathan Rommel Noufi 235CF Times Cited:710 Cited References Count:15 , 235CF Times Cited:710 Cited References Count:15 , The following values have no corresponding Zotero field: Author Address: Contreras, MA NREL, MS 3211,1617 Cole Blvd, Golden, CO 80401 USA NREL, MS 3211,1617 Cole Blvd, Golden, CO 80401 USA NREL, Golden, CO 80401 USA Colorado State Univ, Dept Phys, PV Lab, Ft Collins, CO 80523 USA , The following values have no corresponding Zotero field: Author Address: Contreras, MA NREL, MS 3211,1617 Cole Blvd, Golden, CO 80401 USA NREL, MS 3211,1617 Cole Blvd, Golden, CO 80401 USA NREL, Golden, CO 80401 USA Colorado State Univ, Dept Phys, PV Lab, Ft Collins, CO 80523 USA Understanding the defect physics in polycrystalline photovoltaic materials Article Dec 2007 PHYSICA B C.-S. Jiang Mowafak Al-Jassim We use the combination of high-resolution electron microscopy and density-functional theory to study the atomic structure and electronic effects of structural defects, such as lamellar twins, stacking faults, and double-positioning twin boundaries in polycrystalline photovoltaic materials such as Si, CdTe, and CuInSe2. We find that individual lamellar twins and stacking faults do not create deep levels in all these materials. However, areas with high density of these defects can form buried wurtzite layers that introduce a barrier to the majority carriers. Double-positioning twin boundaries, which contain dislocation cores, create deep levels in Si and CdTe. Surprisingly, however, they do not create deep levels in CuInSe2. These results may explain the fact that Si and CdTe solar cells usually require special passivation, whereas CuInSe2 solar cells do not. Our further study on the passivation effects indicates that grain boundaries in Si cannot be passivated completely by H alone. On the other hand, grain boundaries in CdTe can be passivated well by Cl and I. Influence of Na and H2O on the surface properties of Cu(In,Ga)Se2 thin films Article Sep 1997 Clemens Heske G. Richter Zhonghui Chen Franz Karg The influence of humidity on the electronic structure of Na-containing polycrystalline Cu(In,Ga)Se2 thin films on soda-lime glass substrates has been investigated by x-ray and UV photoemission as well as by Auger electron spectroscopy. Different interactions between coadsorbed Na, H2O, and the Cu(In,Ga)Se2 surface are revealed at low temperatures and upon annealing at room temperature. Both, reversible and irreversible interactions such as a H2O-induced reduction of the Na surface content and the formation of a Na–O–Cu(In,Ga)Se2 complex are observed. Our findings can be correlated with the influence of ambient conditions on Cu(In,Ga)Se2 solar cell adsorbers and demonstrate the importance of adequate encapsulation of Na-containing Cu(In,Ga)Se2-based thin-film solar cells. Prospects for in situ junction formation in CuInSe2 based solar cells Article Jul 1998 SOL ENERG MAT SOL C Kannan Ramanathan Rommel Noufi In this paper we describe our research efforts directed towards the understanding of the CdS/CuInGaSe2 junctions and, specifically, the interaction of the chemical bath with the CuInGaSe2. Information gained from these studies has been used to develop a set of criteria for forming junctions without the need for chemical bath deposition or CdS. Our approach differs from many others previously used “alternative buffer layer” methods which appear to be somewhat problematic in implementation as well as in the quality of the results. This “buffer-free” technology has resulted in a 13.5% efficiency cell. Mono- and tri-crystalline Si for PV production Article Apr 2002 SOL ENERG MAT SOL C A. L. Endrös Crystalline silicon wafers are by far the dominant absorber materials for today's production of solar cells and modules due to their good price/performance relation and their proven environmental stability. These wafers are mainly produced either by a solar-optimized Czochralski (Cz)-growth method yielding crystalline silicon with low defect density (c-Si) or by a directional solidification or a ribbon growth method yielding large grained multi-crystalline (mc-Si) wafers with higher defect density. To further improve the price/performance relation of Cz solar cells, tri-crystalline silicon (tri-Si) is being developed as a high-quality wafer material that combines both the high diffusion length of minority carriers of up to 1300 μm of c-Si and the productivity of mc-Si. More than 1000 μm LID free diffusion length could be reached with specially doped tri-crystals. Due to an increased mechanical stability tri-Si allows both quasi-continuous pulling and thin slicing with higher mechanical yields. This paper reviews the structural, electronic, and mechanical properties of tri-crystalline silicon wafers with respect to c-Si wafers for solar applications. Actual non-textured solar cells processed with a simple cost effective fabrication process exhibit the same cell efficiencies up to 15.9% for both tri-silicon and mono-silicon wafers. With an improved process, up to 18% cell efficiency can be obtained with textured mono-Si. 2D-numerical analysis and optimum design of thin film silicon solar cells Article Jan 2001 SOL ENERG MAT SOL C Tsutomu Yamazaki Akihiro Nagatani Yoshihiro Hamakawa Takuya Matsui Device modeling for p–i–n junction μc-Si basis thin film polycrystalline Si solar cells has been examined with a simple model of columnar grain structure and its boundary condition utilizing two-dimensional device simulator. As the simulation results of solar cell characteristics show, open-circuit voltage (Voc) and curve fill factor (FF) considerably depend on those structural parameters, while short-circuit current density (Jsc) is comparatively stable by courtesy of homogeneous built-in electric field in the i layer. It has also been found that conversion efficiency over 12% could be expected with 1 μm grain size and well-passivated condition with 3 μm thick i-layer. We grow epitaxial silicon films on (100) Si wafers at low temperature (<400 °C), from silane radicals, to understand the mechanisms of sudden epitaxy breakdown and simultaneous growth of amorphous and crystalline silicon phases. Surface roughness is below 0.7 nm before breakdown and below 1 nm in epitaxial regions persisting after breakdown, in contrast to the roughness-induced breakdown observed in molecular beam epitaxy from atomic Si. Spherical caps of hydrogenated amorphous Si (a-Si:H) breakdown cones protrude above the crystal surface, with each sphere centered on its cone apex. This means that the a-Si:H grows isotropically from impinging radicals with low surface mobility and that the a-Si:H growth rate is higher than the epitaxial growth rate. Similar physical mechanisms likely apply to nanocrystalline silicon film growth. Probing Carrier Depletion on Grain Boundaries in Polycrystalline Si Thin Films by Scanning Capacitance Microscopy Mater Res Soc Symp Proc Grain boundaries (GBs) in polycrystalline Si thin-film solar cells are believed to limit the photovoltaic efficiencies. In this paper, we report on a nanometer-resolution measurement of the carrier depletion at the GBs, using scanning capacitance microscopy (SCM). The SCM images exhibit the following features: (1) Carrier concentrations are lower at locations around the GBs than on center regions of the grains; (2) The depletion width at the GBs varies considerably, between 0 and 100 nm, depending on individual GBs; (3) Intra-grain carrier depletion was also observed at point and line defects; and (4) The faceted features that were observed on the topography of the as-grown film surface appeared on the SCM images even after the film surface was polished flat. The direct measurement of the carrier depletion on the GBs demonstrates that the GBs in Si thin films indeed create charged gap states. The nonuniformity of the carrier depletions suggests that the gap states depend on specific GB structures, which should relate directly to the grain orientations and grain facets adjacent to the GB. The depletion around the intragrain defects indicates that the defects are charged and can be recombination centers, and thus, harmful to device performance. This paper reports the first step of our studies toward understanding the relationships between the electronic and structural properties on specific GBs. We report on direct measurements of the built-in electrical potential in III-V semiconductor-based solar cell devices by using scanning Kelvin probe microscopy. Potential profiles on cross sections of the devices were measured quantitatively and spatially resolved in open and short circuits, under and without illuminations, with selective photon energies matching band gaps of the junctions. The measurements provide valuable information about the electrical properties of the devices, and are useful for understanding the performance of solar cells. On a GaInP2-single junction cell, two potential features were measured and were assigned to the p-n junction and the potential barrier at the interface between the GaInP2 base layer and the GaAs substrate. The potential on the p-n junction is photoactive, and that on the GaInP2/GaAs interface is photoinactive. On a GaInP2/GaAs tandem cell, two potential features were measured near the top and the bottom p-n junctions. When the sample was illuminated by light with photon energies larger than the band gap of the top junction, in the condition of short circuit, a potential peak appeared on the boundary between the bottom cell and tunnel junction due to charge accumulations on the bottom junction. Significant improvement in silicon chemical vapor deposition epitaxy above the surface dehydrogenation temperature Paul Stradins Charles Teplin We observe an order of magnitude increase in both the rate and achievable thickness of epitaxy by hot-wire chemical vapor deposition at temperatures above the dehydrogenation temperature of the silicon surface. We deposit silicon films on (100) silicon at 110 nm/min at substrate temperatures between 520 and 645 °C. At the glass compatible temperature of 610 °C, we observe phase-pure epitaxial growth of more than 10 mum, as observed by x-ray diffraction and transmission electron microscopy, in contrast to the mixed-phase breakdown seen at lower temperatures. In 610 °C films thicker than 4 mum, a stable (100)-epitaxial growth mode produces regular surface pyramids with vicinal facets. On the low-energy (111) face of Si, more than 2 mum of epitaxial Si were grown at 645 °C, but some twinning is observed. The temperature of this new growth regime suggests that rapid dehydrogenation of the growing surface is critical for silicon epitaxy by CVD. Local current flow in amorphous and nanocrystalline mixed-phase silicon solar cells Local current flow in amorphous and nanocrystalline mixed-phase n-i-p silicon solar cells is measured using conductive atomic force microscopy (C-AFM) and correlated to the material structure from Raman measurement. The C-AFM images show that the current is very low over the entire surface of the fully amorphous region. High current spikes are observed in the mixed-phase region, where the current spike corresponds to aggregations of nanocrystallites. The size of the nanocrystalline aggregations is on the order of a half micrometer in diameter. The density of the current spike increases from the mixed phase to the heavily nanocrystalline regions. A thick amorphous silicon buffer layer inserted between the p and i layers significantly reduced the magnitude of the current spike. The C-AFM measurements suggest that the mixed-phase cells can be considered as a two-phase parallel-connected diode structure, consistent with our previously proposed model. Effects of Bismuth on Wide-Depletion-Width GaInNAs Solar Cells J Vac Sci Tech B Aaron J. Ptak R. C. Reedy C.-S. Jiang GaInNAs solar cells could be useful in next-generation multijunction solar cells if issues surrounding low photocurrents and photovoltages are surmounted. Wide-depletion-width devices generate significant photocurrent using a p-i-n structure grown by molecular beam epitaxy, but these depletion widths are only realized in a region of parameter space that leads to rough surface morphologies. Here, bismuth is explored as a surfactant for the growth of GaInNAs solar cells. Very low fluxes of Bi are effective at maintaining smooth surfaces, even at high growth temperatures and In contents. However, Bi also increases the net donor concentration in these materials, manifested in our n-on-p device structures as a pn-junction that moves deeper into the base layer with increasing Bi fluxes. Quantum efficiency modeling and scanning kelvin probe microscopy measurements confirm the type conversion of the base layer from p type to n type. Bi incorporation in GaAsBi samples shows signs of surface segregation, leading to a finite buildup time, and this effect may lead to slow changes in the electrical properties of the GaInNAs(Bi) devices. Bi also appears to create a defect level, although this defect level is not deleterious enough to increase the dark current in the devices. Theoretical analysis of the effect of conduction band ofset of window/CIS layers on performance of CIS solar cells using device simulation SOL ENERG MAT SOL C Takashi Minemoto Takuya Matsui Hideyuki Takakura One of the most important factors of CdS leading to high performance in Cu(In,Ga)Se2 (CIGS) solar cells is appropriation of the conduction band offset of CdS/CIGS layers. However, it is not clearly explained. In this study, device modeling and simulation were conducted to explain the effect of conduction band offset of window/CIGS layers on performance of CIGS solar cells. As a result of calculation, excellent performance can be obtained when the conduction band of window layer positions higher by 0–0.4eV than that of CIGS. Microscopic characterization of individual grain boundaries in Cu-III–VI 2 chalcopyrites THIN SOLID FILMS Sascha Sadewasser The role of grain boundaries in polycrystalline Cu-III–VI2 absorber material for thin film photovoltaics has not been fully understood and is currently under discussion. Recently, intensive efforts have been devoted to the characterization of the properties of individual grain boundaries using microscopic techniques, including Kelvin probe force microscopy (KPFM). KPFM provides local electronic information by measuring the surface potential in addition to the topography. We introduce the KPFM method and present simulations assessing the technique's limitations with respect to spatial resolution regarding the measurement of grain boundary properties. KPFM studies of individual GBs in the Cu(In,Ga)Se2 materials system are reviewed and critically discussed, considering also results from other microscopic characterization techniques. Large grain polycrystalline Si thin films by nucleation-controlled chemical vapor deposition using intermittent source gas supply Tsutomu Yamazaki Yukiharu Uraoka Takashi Fuyuki Polycrystalline Si (poly-Si) thin films with large grain size have been deposited by nucleation control with intermittent source gas supply using atmospheric pressure chemical vapor deposition. The grain size can be controlled in the range from 3.1 to 31 μm. The Hall mobility of poly-Si thin films increased to 69.9 cm2/V s for a grain size of 15 μm after hydrogenation treatment. The grain boundaries in this film have a weak influence on the majority carrier transport due to effectively passivated grain boundaries and larger grain size. The photovoltaic performance of poly-Si thin film solar cells, especially the open circuit voltage, was also improved with the increase of the grain size. Jan 2005 THIN SOLID FILMS C.-S. Jiang Helio Moutinho M. J. Romero Qing Wang We report on a direct measurement of the spatial distribution of electrical potential on cross sections of hydrogenated amorphous silicon (a-Si:H) n–i–p solar cells using scanning Kelvin probe microscopy. We found that most voltage has dropped near the p/i interface up to ∼500 nm into the i-layer, but that the voltage flattens out near the n/i region for a device with i-layer thickness of ∼800 nm. However, the potential distributes approximate-linearly on the entire i-layer for a device with i-layer of ∼250 nm. The potential measurements suggest that the i-layer is slightly n-type (6–13×1015/cm3), and the depletion width is ∼350–500 nm at the i/p interface. This depletion width provides a direct experimental evidence for the device design criteria that a-Si:H solar cells should be thinner than ∼500 nm, in the point view of electrical potential. Influence of sodium on the growth of polycrystalline Cu(In,Ga)Se 2 thin films Article Feb 2000 THIN SOLID FILMS D. Braunger Dimitrios Hariskos G. Bilger Hans-Werner Schock We investigate the influence of Na on the growth of Cu(In,Ga)Se2 thin-films by three model experiments. First, we examine the influence of Na on the Se activity during selenisation of Mo films on soda-lime and borosilicate glass after growth and after thermal treatments. Second, we analyse the location and possible binding partners of Na in polycrystalline Cu(In,Ga)Se2 prior to and after air-exposure. The final experiment focuses on the identification of the chemical state of Na on the surface of as grown and air-exposed films. Our experiments demonstrate that Na influences the growth of CIGS Cu(In,Ga)Se2 due to its interaction with Se. In non-air-exposed films Na is mainly localised in the form of sodium-polyselenides (Na2Sex) at the grain boundaries. We conclude that Na2Sex acts as Se-reservoir during film formation and oxidation. Polycrystalline Silicon For Integrated Circuit Applications Ted Kamins Surfactant enhanced growth of GaNAs and InGaNAs using bismuth Apr 2003 J CRYST GROWTH S. Tixier M. Adamcyk Erin C. Young Tom Tiedje Dilute nitride GaNAs thin films and InGaNAs single quantum wells (QWs) have been grown by molecular beam epitaxy with a concurrent bismuth flux. Bi does not incorporate into the films and acts as a surfactant. Atomic force microscopy images reveal that, at sufficiently high bismuth flux, step flow growth occurs in GaN0.004As0.996 even at substrate temperatures as low as 460°C. This results in an order of magnitude decrease in the surface roughness. A similar smoothing effect is obtained when growing GaAs and AlGaAs thin films with bismuth. Furthermore, Bi is found to enhance the incorporation of nitrogen into GaNAs. The peak photoluminescence intensity from an In0.26Ga0.74N0.011As0.989 QW is increased by more than a factor of two with the surfactant. We conclude that Bi reduces the incorporation of defects and/or impurities in the dilute GaNAs based alloys. Analysis of p-n Junction Profiles of Polycrystalline Silicon Thin-Film Solar Cells by Electron-Beam-Induced Current Technique Article Apr 2006 Tsutomu Yamazaki Yukiharu Uraoka Takashi Fuyuki The correlation between the p-n junction profiles and photovoltaic performance of polycrystalline silicon (poly-Si) thin-film solar cells deposited by atmospheric chemical vapor deposition is discussed. The p-n junctions were formed by two methods applicable to poly-Si thin-film solar cells, i.e., a conventional solid-phase diffusion process and a low temperature deposition method. The junction profiles were directly observed by a high-resolution cross-sectional electron-beam-induced current technique. The p-n junctions perpendicular to the substrate due to the preferential diffusion of phosphorous atoms along grain boundaries were observed for the high-temperature diffusion process. The perpendicular p-n junction induced the high carrier collection probability through the grain boundaries. The effective carrier diffusion length linearly increased with the p-n junction depth along grain boundaries. In this case, however, the shunt resistance deteriorated due to the carrier recombination through defects at the grain boundaries. The low temperature deposition emitter could suppress the preferential diffusion and then the recombination process through the grain boundaries. Dec 2001 Yasuaki Ishikawa Yukie Yamamoto Tomoaki Hatayama Takashi Fuyuki In order to realize high efficiency thin film polycrystalline silicon (poly-Si) solar cells, a novel method of quasi three-dimensional simulation using a cylindrical coordinate system was proposed. Optimum design of cell configuration and analysis of device performance have been demonstrated. In this simulation we used realistic physical values for parameters, such as interface recombination velocity at grain boundaries and diffusion length of minority carriers. Interface recombination velocity at grain boundaries (which has a strong effect on cell performance) should be less than 103 cm/s to obtain high cell performance. Even at a relatively short diffusion length of 50 mum, high efficiency of more than 16-18% can be expected at a film thickness of 5-20 mum when grain size is 10 mum, which is appropriate for the utilization of solar grade Si of reasonable material quality. Observation of Strong Ordering in Ga x In 1 − x P alloy semiconductors Akiko Gomyo Tohru Suzuki Sumio Iijima Extremely strong superstructure reflections have been observed for the first time by transmission electron microscopy for (110) edge-on Ga0.5In0.5P crystals lattice matched to GaAs. Growth was carried out by metal-organic vapor-phase epitaxy. A monolayer superlattice (SL) of (Ga-rich plane) (In-rich plane) . . . is formed along the [1¯11] and [11¯1] directions. The substrates are found to be playing a vital role in the SL formation. Band-gap energy reduction (~=80 meV) has been observed for layers with these SL's. Influence of boron doping on roughness microcrystalline silicon May 2008 J NON-CRYST SOLIDS W. Yoshida Yasushi Sobajima H. Okamoto We have studied roughness of boron-doped microcrystalline Si (μc-Si) surfaces with an emphasis on the influence of heavy doping. μc-Si films were prepared using plasma-enhanced chemical vapor deposition (PECVD) with different boron concentrations in gas phase from 0% to 2%. Growth-induced roughening of μc-Si surfaces was monitored ex situ using an atomic force microscope (AFM). With an increase in the deposition time, the surface width (rms roughness), w, of undoped μc-Si surface exhibited usual behaviors; first, (a) w increased, (b) slightly dropped, (c) rose again, and then (d) gradually increased. In the case of B-doped μc-Si, w differently behaved; (a) w increased very soon, (b) slightly dropped, (a′) rose again, (b′) slightly dropped again, (c) rose, and finally (d) gradually increased. The quick increase in w indicates that boron doping promotes the nucleation, and the repeated nucleation is responsible for the behavior (a′)–(b′). Additionally, the nucleation density, that was derived using the lateral correlation length of surface heights, monotonically increased with an increase in the boron concentration. The effects of boron doping are discussed with the catalytic effects and the formation of the surface-covering layer. Nanopatterning of Si/SiGe electrical devices by atomic force microscopy oxidation Oct 2002 APPL PHYS LETT Xiang-Zheng Bo L. P. Rokhinson Haizhou Yin James C. Sturm Two nanopatterning methods for silicon/silicon-germanium (Si/SiGe) heterostructures are demonstrated: (1) direct atomic force microscopy (AFM) oxidation on SiGe layers and (2) AFM oxidation on silicon followed by selective wet etching of SiGe. When directly oxidizing SiGe alloys, minimum linewidths of 20 nm were achieved by adjusting the bias voltage of the AFM tip. By AFM oxidation and selective wet etching, a 10-nm-thick conducting SiGe layer was patterned to form features under approx50 nm. Fabricated SiGe quantum dots with side gates exhibited Coulomb blockade oscillation. Handbook series on semiconductor parameters Jan 1996 M Levinstein Sergey L. Rumyantsev Growth of multicrystalline Si with controlled grain boundary configuration by the floating zone technique The floating zone technique was employed to grow multicrystalline Si with controlled grain boundary configuration. Purposely designed bi-crystals were utilized as seed crystals to investigate the effect of the tilt angle from the perfect twin boundary on the growth behavior. When the growth was initiated from a bi-crystal with a Σ3 twin boundary, no particular change took place on the grain boundary configuration during growth. On the other hand, the decrease of the tilt angle during growth was observed when the growth was initiated from a bi-crystal with a tilted boundary from Σ3. This was accompanied by the appearance of new crystal grains. The reduction of the total interface energy would be a possible driving mechanism for this phenomenon. Grain Enlargement of Polycrystalline Silicon by Multipulse Excimer Laser Annealing: Role of Hydrogen The role of hydrogen introduced into melt-Si during excimer laser annealing (ELA) is examined from the viewpoint of grain enlargement. An amorphous silicon (a-Si)/SiN/quartz glass structure is successfully prepared by a catalytic chemical vapor deposition (Cat-CVD) method for a SiN film, in which the hydrogen concentration of the SiN film is controlled. The grain size increases as the hydrogen concentration decreases, and it partially exceeds 2 mum when the hydrogen concentration of the SiN film is fixed at 2.3 at. %. The relationship between defects at grain boundary and hydrogen is also considered. Consolidated tables showing an extensive listing of the highest independently confirmed efficiencies for solar cells and modules are presented. Guidelines for inclusion of results into these tables are outlined and new entries since July 2009 are reviewed. Current routes in hydrogenated microcrystalline silicon The numerous studies of electrical transport in undoped hydrogenated microcrystalline silicon (μc‐Si:H) failed so far to establish an agreement of where does the current flow in this heterogeneous system. Here we present a comprehensive local probe investigation that solves this intriguing question and sets up a self consistent picture of the conduction mechanisms and routes in this system. The corresponding significance regarding the emerging field of percolation in semiconductor composites and the related photovoltaic applications are discussed. We report on a method to quantitatively measure the local energy distribution of surface states density within the band gap of semiconductors using Kelvin probe force microscopy. The method is based on measuring the contact potential difference of a cross-sectional pn junction; as the tip scans the junction surface, the surface states population changes, thus modifying the measured local surface band bending. The energy distribution is then obtained by fitting the calculated surface potential to the measured contact potential difference. The method is applied to p++n and n++p Si (110) surfaces and p++n InP (110) surfaces measured under different environments. The results are compared to data obtained by theoretical models and other experimental methods. Determination of energy levels of surface states in GaAs metal–semiconductor field-effect transistor using deep-level transient spectroscopy The energy levels of surface states at the surface of GaAs were determined through capacitance deep-level transient spectroscopy of GaAs metal–semiconductor field-effect transistor with large gate periphery. Two types of hole-like traps are observed in the spectra. These originate from the surface states at the ungated regions between gate and source/drain electrodes. The activation energies of both surface states are determined to be 0.65±0.07 and 0.88±0.04 eV, which agree well with the energy levels of AsGa+ and AsGa++ within band gap of GaAs, responsible for the Fermi level pinning at the surface. © 1999 American Institute of Physics. Changes in the dominant recombination mechanisms of polycrystalline Cu(In,Ga)Se2 occurring during growth The changes in dominant recombination mechanisms of Cu(In,Ga)Se2 thin films grown from (In,Ga)2Se3 precursors are investigated using energy- and time-resolved photoluminescence. The results are analyzed with a rate-equation analysis and correlated with n/p diode measurements on processed devices. The experimental results quantify a change in the dominant radiative process and an improvement in the underlying material quality. These observations support a growth model incorporating changes in the dominant defect states and recombination mechanisms during this final stage of the growth process. Additionally, evidence is presented that supports the passivation of near-surface recombination sites by CdS. © 2003 American Institute of Physics. Lateral electron transport in Cu(In,Ga)Se2 investigated by electro-assisted scanning tunneling microscopy Chun-Sheng Jiang We investigate the lateral electron transport across grain boundaries in Cu(In,Ga)Se-2 (CIGS) by a combination of scanning tunneling microscopy (STM) with the excitation provided by the electron beam in electron microscopy-or electro-assisted STM. Using this method, we report evidence for a significant barrier for electron diffusion across grain boundaries in CuGaSe2 (CGS), which is not present in CuInSe2 (CIS). Finally, we discuss the effects of gallium addition. (C) 2005 American Institute of Physics. Scanning probe microscopy polarization experiments with polycrystalline Ce0.8Gd0.2−xPrxO2−δ and Ce0.... Kerstin Neuhaus Falk Schulze-Küppers Stefan Baumann [...] Hans-Dieter Wiemhöfer Whereas the defect chemistry of acceptor doped ceria is well understood at temperatures above 300 °C, the information on polarization and mixed ionic electronic conduction at much lower temperatures is by far less understood. By using an AFM probe first as an electron conducting nanoelectrode in contact to a doped ceria sample surface, a constant voltage pulse was applied to achieve a local ... [Show full abstract] polarization with distinctly changed redox state and defect concentrations. A subsequent mapping experiment used the AFM as Kelvin probe to scan local work function distributions on the sample surfaces in air. In this way, reversible room temperature changes could be monitored in an extended region around pre-polarized surface locations. For a ceria single crystal (acceptor doped with 20 mol% Y), a large distance zone with time-dependent surface polarization was well visible with clearly monitored reversible changes upon initially applied positive and negative bias. By comparing results for Ce0.8Y0.2O1.9 single crystals and a series of co-doped ceramic samples with composition Ce0.8Gd0.2-xPrxO2-δ (x=0.2, 0.09, and 0.01), the role of grain boundaries as well as the effect of an increasing concentration of the redox active dopant Pr3+/4+ could be studied with regard to the local, near-surface defect mobility and concomitant redox processes. Scanning Probes of Nonlinear Properties in Complex Materials June 2004 · Japanese Journal of Applied Physics Rui Shao D. A. Bonnell Nonlinear materials have found wide applications in electronic devices. Of special importance and interest are those with varistor-type grain boundaries and those displaying large piezoelectric and electrostrictive responses to external fields. To access the local properties of these materials, we have designed two new contact mode scanning probe techniques. The first technique referred to as ... [Show full abstract] nanoimpedance microscopy/spectroscopy, which incorporates impedance spectroscopy with a conducting AFM tip as an electrode, has been demonstrated on measuring local grain boundary properties of a ZnO varistor, as well as the contact quality between AFM tip and a ferroelectric sample. The other technique referred to as second harmonic piezoresponse force microscopy developed to measure the electrosctriction has been demonstrated on ferroelectric polymer thin film. A theoretical model has been presented to describe the contrast formation of the second harmonic as well as the first harmonic (piezoresponse) images and to explain the observed hysteretic field dependence of the second harmonic amplitude signal. Thermal imaging of electronic materials and devices using the Atomic Force Microscope June 1993 · Proceedings of SPIE - The International Society for Optical Engineering Arun Majumdar M. Chandrachood We have developed a new and simple technique of thermal imaging with sub-micron spatial resolution using the atomic force microscope (AFM). By using a thermocouple as an AFM tip, we can simultaneously observe the topography and the temperature field of material surfaces. The method is particularly unique for application of biased electronic devices or interconnects where there could be different ... [Show full abstract] materials and potential variations on a scan surface. Application to a n-GaAs MESFET has revealed hot spots under the drain-side of the gate. Thermal images of a biased polycrystalline Al-Cu via structure show the grain boundaries to be hotter than within the grain suggesting higher electron scattering rates. We have also observed the effects of current crowding in generating hot spots in the via structure. An error analysis showed that the difference between the measured temperature and the estimated actual device temperature is about 4 percent. Reconstruction of dopant vertical position from Kelvin probe force microscope images Krzysztof Tyszka Ryszard Jablonski In novel nano-scale electronic devices the number and location of individual dopant atoms within a device determine its characteristics. Therefore, precise control over these parameters is required. In this paper we describe Kelvin Probe Force Microscope (KPFM) designed for dopant detection. We propose a method for reconstruction of dopant position by using comparison between KPFM images with ... [Show full abstract] surface potential simulation based on Thomas- Fermi approximation. This is the first step to allow 3D reconstruction of manydopant arrangements from 2D KPFM images. C-AFM and KPFM approach to investigate the electrical properties of single grain boundaries in ZnO v... March 2013 · Proceedings of SPIE - The International Society for Optical Engineering Andreas Nevosad Michael Hofstätter Manfred Wiessner [...] Christian Teichert Here, two Praseodymium doped ZnO multilayer varistor (MLV) samples with Ag/Pd electrodes were macroscopically and microscopically investigated. Since the varistor effect is a grain boundary effect, the electrical properties of individual ZnO grain boundaries were especial subject of this study. Both samples were cross sectioned for the AFM based investigation. Besides conductive atomic force ... [Show full abstract] microscopy (C-AFM), Kelvin Probe Force Microscopy (KPFM) and KPFM with a voltage applied between the varistor electrodes, so called scanning surface potential microscopy (SSPM), have been employed. Both samples were mapped with electron backscatter diffraction (EBSD) after the AFM based investigations. In C-AFM, a high conductivity of the ZnO grains and a high resistivity of the grain boundaries was proven. With SSPM, individual grain boundaries with an asymmetric electrical behavior were identified. These grain boundaries are causing asymmetric current paths and therefore can cause asymmetric device behavior. Grain Boundary Engineering of Halide Perovskite CH 3 NH 3 PbI 3 Solar Cells with Photochemically-Act... Nastaran Faraji Chuanjiang Qin Toshinori Matsushima [...] Jan Seidel In this study, we investigate the nanoscale effects of photochemically-active additives of benzoquinone (BQ), hydroquinone (HQ), and tetracyanoquinodimethane (TCNQ) on grain boundaries in CH3NH3PbI3 solar cells. We employ scanning probe microscopy under light illumination, in particular Kelvin probe force microscopy, to study surface potential changes under laser light illumination. The recently ... [Show full abstract] found improvement in efficiency of BQ added solar cells can be clearly seen in vanishing contact potential differences at grain boundaries under illumination, rendering the material more uniform in solar cell operating conditions. These effects are observed for BQ, but not for HQ and TCNQ. Our findings shed light onto halide perovskite materials and functional additive design for improved solar cell performance. Spatial variation of wear and electrical properties across wrinkles in chemical vapor deposition gra... Borislav Vasić Amaia Zurutuza Radoš Gajić Chemical vapor deposition (CVD) is an effective and simple method for production of large area graphene needed for technological applications. However, CVD graphene contains a network of out of plane deformations - graphene wrinkles. Using atomic force microscopy (AFM) based methods, we find a significant change of graphene wear and electrical properties across the wrinkles. It is shown that ... [Show full abstract] graphene tearing by an AFM tip starts exactly from the wrinkles. Kelvin probe force microscopy shows that the surface potential of graphene is inhomogeneous, with domains within closed wrinkles having the surface potential different from the surrounding graphene. Conductive AFM shows that the electrical current drops exactly along wrinkles. Therefore, wrinkles decrease wear resistivity of graphene, they act as potential barriers for charge carriers leading to their localization, and lead to charge carrier scattering and increased contact resistance. Doping of Grain Boundaries in diF TESADT Transistors Cortney Bougher Shawn Huston Jeremy W. Ward [...] We utilize Atomic Force Microscopy (AFM) and Kelvin Probe Force Microscopy (KPFM) to characterize the dynamics of electronic transport across 2,8-difluoro-5,11-triethylsilylethynyl anthradithiophene (diF TESADT) grain boundaries. We show that the morphology of grain boundaries and the adsorption of atmospheric dopants at these local boundaries have a direct impact on the electrical behavior of ... [Show full abstract] diF TESADT in thin film transistor (TFT) devices. Device voltage drops at grain boundaries are characterized as a function of both atmospheric dopants and transition time between dopants. The morphology, including crystallization and packing motifs, of diF TESADT grown on thermally grown SiO2 will be discussed and related to other semiconducting small organic molecules. This work will be put in the context of other, recent advances in small molecule organics. Direct Measurement of Electronic Band Structure in Single Quantum Dots of Metal Chalcogenide Composi... Daniele Benetti Daling Cui Haiguang Zhao [...] Alberto Vomiero Metal chalcogenide quantum dots (QDs) are among the most promising materials as light harvesters in all‐inorganic systems for applications in solar cells and production of solar fuels. The electronic band structure of composite QDs formed by lead and cadmium chalcogenides directly grafted on highly oriented pyrolytic graphite surfaces through successive ionic layer absorption and reaction is ... [Show full abstract] investigated. Atomic force microscopy and Kelvin probe force microscopy (KPFM) are applied to investigate PbS, CdS, and PbS/CdS QD systems. The variation of the surface potential of individual QDs is measured, investigating the evolution of the electronic band structure as a function of QD size and composition. A shift of the Fermi level toward more negative values occurs when QD size is increased. The shift is more pronounced in CdS than in PbS, while the composite PbS/CdS exhibits an intermediate behavior. The calculated shift is in good agreement with the experiments. These results highlight the ability of KPFM to directly measure the electronic band structure in individual QDs of metal chalcogenide composites. This feature regulates charge dynamics in composite systems, thereby affecting device performance. This work provides valuable insights for applications in several fields, in which charge injection plays a major role. The electrical properties of individual quantum dots (QDs) with different size and composition are studied with Kelvin Probe Force Microscopy (KPFM). The KPFM is able to follow the variation of the Fermi level inside a single QD and to distinguish the different materials from the electronic point of view, without being influenced by the morphology of the sample. Influence of Grain Boundaries on Dissolution Behavior of a Biomedical CoCrMo Alloy: In-Situ Electroc... Eleonora Bettini Christofer Leygraf Changjian Lin [...] Jinshan Pan	https://www.researchgate.net/publication/251113042_Microscopic_Electrical_Characterization_of_Inorganic_Semiconductor-Based_Solar_Cell_Materials_and_Devices_Using_AFM-Based_Techniques
The impact of Neonatal Resuscitation Program courses on mortality and morbidity of newborn infants with perinatal asphyxia \| Request PDF Request PDF \| The impact of Neonatal Resuscitation Program courses on mortality and morbidity of newborn infants with perinatal asphyxia \| Objective. Neonatal care provided within the first few minutes of life plays a major role in the reduction of neonatal morbidity and mortality.... \| Find, read and cite all the research you need on ResearchGate The impact of Neonatal Resuscitation Program courses on mortality and morbidity of newborn infants with perinatal asphyxia Abstract Objective. Neonatal care provided within the first few minutes of life plays a major role in the reduction of neonatal morbidity and mortality. Neonatal Resuscitation Program (NRP) courses had been held since 1996. The aim of this study was to evaluate the impact of the NRP on morbidity and mortality of newborn infants with perinatal asphyxia. Methods. This retrospective study comprised newborn infants who were born in hospitals at Trakya region of Turkey during the last 3 years and were diagnosed as perinatal asphyxia and were referred to our Neonatal Unit. Those patients who were referred before NRP course (pretraining period) were designated as Group 1, those who were referred after the first NRP course (transition period) as Group 2, and those who were referred after the second NRP course (post-training period) as Group 3. Chart review was performed with regard to gestational age, birth weight, Apgar scores, resuscitation type, stage of hypoxic ischemic encephalopathy (HIE), existence of meconium aspiration syndrome (MAS), progress of the disease, duration of hospitalization. Results. The study comprised 66 patients; 35 in Group 1, 18 in Group 2 and 13 in Group 3. The number of cases who had not been resuscitated was 10 in the pretraining period, 3 in the transition period and 1 in the post-training period which decreased significantly. The first minute Apgar scores in three groups were as follows; 2.08 ± 1.2, 2.2 ± 1.1 and 3.7 ± 1.4, and this increase was statistically significant. The fifth minute Apgar scores also increased from 5.43 ± 1.5 in the pretraining period to 6.5 ± 1.9 in the post-training period, but this increase was not statistically significant. The number of patients with Stage 1 and 2 HIE decreased more in Group 3 (n = 11 in Stage 1 HIE, n = 17 in Stage 2 HIE) compared to those in Group 1 (n = 7 in Stage 1 HIE, n = 5 in Stage 2 HIE) but the difference was not statistically significant. The duration of hospitalization decreased in post-training period (15.1 ± 10.3 days in pretraining period, 12.0 ± 8.9 days in transition period, 6.1 ± 1.2 days in post-training period). Conclusions. After NRP courses, the number of patients with perinatal asphyxia and with no resuscitation and also the duration of hospitalization decreased significantly, whereas the first minute Apgar scores increased significantly. Request full-text PDF ... Didactic training using the Neonatal resuscitation Program (NRP) of the American Association of Paediatrics (AAP) has been recognised as an avenue for the acquisition of resuscitation knowledge and skills by health workers who attend mothers and babies at delivery 9 . Various studies have shown that neonatal resuscitation training can be followed immediately by significant improvement in health workers practices 9, 10, 11,12 . However little attention has been paid to furnishing health workers with these skills inspite of the widespread inappropriate, ineffective forms of practice 13,14 . ... ... This trend has also been observed in earlier studies 15,16 . Opiyo et al also observed an improvement in cognitive knowledge and skills over a seven week period and a reduction in harmful resuscitation practices between the trained and untrained health workers 10 . ... Oloyede IP Effectiveness of didactic training on the cognitive knowledge of health professionals on neonatal resuscitation in southern Nigeria Article Full-text available Mar 2021 Niger J Paediatr E.E. Nyong Iso Oloyede PA Udo Background: Nigeria has a high neonatal mortality rate. Most of these deaths can be prevented by providing adequate training for health providers with available and functional basic resuscitation equipments. Our aim was to assess the effect of training on the cognitive knowledge of health practitioners on neonatal resuscitation. Method: We conducted neonatal resuscitation trainings for selected health professionals from all the senatorial districts of Akwa Ibom State, based on the Neonatal Resuscitation programme (NRP) of the American Academy of Paediatrics (AAP). The facilitators were trained and certified in the NRP train-the trainers program of the AAP conducted by the Paediatric Association of Nigeria (PAN). Pre-and post-test were organized during the training and the test scores analyzed to assess any improvement in the knowledge of the health professionals on neonatal resuscitation. Results: One hundred and eighty-one health professionals were trained over a two year period. Sixty five (35.9%) were doctors, while 116 (64.9%) were nurses. Physicians had similar pre-test but significantly higher post-test scores compared to the nurses: 46.35±15.34 vs 43.70± 14.51; p=0.34 and 76.1 4±13 .02vs 66.29±15.7; p=0.04 respectively. All the health professionals showed significantly higher post-test scores compared to the pre-test scores; p=0.001 respectively. There was also a negative relationship between the number of practice years and the pre-training scores for the physicians and nurses; spearman rho=-0.18; p=0.45 and-0.43;p=0.003 respectively. Conclusion: Neonatal resuscitation training leads to an improvement in the cognitive knowledge of health practitioners. All health practitioners should be trained irrespective of number of practice years. Further studies are required to assess its long term impact on neonatal mortality. ... 4 Studies have shown that following NRP guidelines decreased neonatal mortality, particularly in moderate-low income countries where resources are scarce. 5, 6 In this context, NRP has also been shown to improve participant knowledge and skills and consequently decrease neonatal mortality and deaths due to birth asphyxia. 6,7 Lebanon is a middle income country where the neonatal mortality rate accounts for 55% of under 5 child mortality and where formal training in neonatal resuscitation was not established until recently. ... ... 5,6 In this context, NRP has also been shown to improve participant knowledge and skills and consequently decrease neonatal mortality and deaths due to birth asphyxia. 6, 7 Lebanon is a middle income country where the neonatal mortality rate accounts for 55% of under 5 child mortality and where formal training in neonatal resuscitation was not established until recently. 8 In 2005-2006, the Lebanese government started planning for the introduction of neonatal resuscitation training at the country level, with the sponsorship of the Latter Day Saint Charities, the humanitarian arm of The Church of Jesus Christ of Latter-day Saints. ... Implementation of the Neonatal Resuscitation Program in a Developing Country Poster May 2013 Lama Charafeddine Walid F. Yassin BACKGROUND: Each year about 99% of the 3.7 million neonatal deaths worldwide occur in developing countries. Minimizing neonatal death is part of the World Health Organization's (WHO) fourth Millennium Development Goal (MDG-4). Teaching neonatal resuscitation is not mandatory in some developing countries. The American Academy of Pediatrics' Neonatal Resuscitation Program (NRP) has been proved to increase the knowledge and practice of medical professionals in infant resuscitation. It is not clear whether the implementation of this course would be sufficient to improve the knowledge in developing countries. OBJECTIVE: Assess the baseline knowledge in neonatal resuscitation and measure the degree of improvement after implementation of the official NRP lectures for learners from different medical backgrounds. DESIGN/METHODS: NRP lectures were given as part of the official course and hands on sessions to nurses, midwives and pediatricians working in urban and rural health care facilities. Participants were asked to complete 36 multiple-choice questions prior to the course without previous knowledge of the material, and after completing the NRP lecture and hands on practice sessions. Descriptive statistical analysis was done using SPSS v.16.0. RESULTS: 109 participants from 22 different hospitals covering all Lebanese governorates completed the course including pre and post-test, and hands on training sessions. Geographic distribution was representative with 30.3% from the capital, 25.7 % from eastern province, 23.8 % from the North, 20.2 % from the South. 54% were nurses, 26.6% physicians, 17.4% midwifes and 1.8% Neonatal intensive care unit nurses. Post test scores showed significant improvement compared with pretest scores (p=0.000) with significant correlation (66.5%, p=0.000) by Wilcoxin signed ranked test. ANOVA Kruskal Wallis test revealed significant difference in mean improvement test scores (M=0.17, SD=0.12) across job titles, hospital types (university vs. community) and locations [Chi square= 7.86; df=2, p=0.02]. CONCLUSIONS: NRP course increased the knowledge of neonatal resuscitation regardless of profession, hospital types and location. This highlights the need for continuing education of neonatal resuscitation on a yearly basis rather than the biannual recommended training. On the other hand this type of training should be mandated at the level of the ministry of health. ... NRP is the developed world's standard of care to prevent death and complications of cerebral palsy due to asphyxia [13], and, when systematically implemented by trained personnel, has the potential to annually prevent 192 000 intrapartumrelated neonatal deaths worldwide and 5-10% of deaths related to preterm complications [14]. Curriculums with components of NRP have significantly improved healthcare 2 International Journal of Pediatrics providers' knowledge, skills, and attitudes in developing countries [15,16] and decreased neonatal mortality in the developing world up to 65.7 percent [10,17, 18] . One study revealed declines in national trends of neonatal mortality over an 8-year period following NRP integration across Malaysia [19]. ... ... Retrospective data revealed 20.9 neonatal deaths per 1000 live births at St. Gabriel's Hospital. Assuming a 44.6% decline [10,17, 18, 20] to 11.6 neonatal deaths per 1000 live births, a two-tail test with 80% statistical power and a 95% confidence interval required at least 2956 subjects in both arms. ... A Neonatal Resuscitation Curriculum in Malawi, Africa: Did It Change In-Hospital Mortality? Article Full-text available Jan 2012 Michael Hole Keely Olmsted Athanase Kiromera Objective. The WHO estimates that 99% of the 3.8 million neonatal deaths occur in developing countries. Neonatal resuscitation training was implemented in Namitete, Malawi. The study's objective was to evaluate the training's impact on hospital staff and neonatal mortality rates. Study Design. Pre-/postcurricular surveys of trainee attitude, knowledge, and skills were analyzed. An observational, longitudinal study of secondary data assessed neonatal mortality. Result. All trainees' (n = 18) outcomes improved, (P = 0.02). Neonatal mortality did not change. There were 3449 births preintervention, 3515 postintervention. Neonatal mortality was 20.9 deaths per 1000 live births preintervention and 21.9/1000 postintervention, (P = 0.86). Conclusion. Short-term pre-/postintervention evaluations frequently reveal positive results, as ours did. Short-term pre- and postintervention evaluations should be interpreted cautiously. Whenever possible, clinical outcomes such as in-hospital mortality should be additionally assessed. More rigorous evaluation strategies should be applied to training programs requiring longitudinal relationships with international community partners. ... Neonatal Resuscitation Program (NRP) guidelines 9 were established by the American Academy of Pediatrics (AAP), American Heart Association (AHA), and International Liaison Committee on Resuscitation (ILCOR) to provide lifesaving care when emergencies occur and improve birth outcomes. [10] [11][12][13] Observational studies of NRP in the hospital setting have identified important gaps in care that need improvement: assessment; initial steps of drying, warming, and stimulating; monitoring and communication of heart rate; and awareness of clinical deterioration. [14][15][16][17] Chart reviews of hospital records have found that paramedics frequently encounter complications due to maternal comorbidities and extreme preterm births. ... Characterization of teamwork and guideline compliance in prehospital neonatal resuscitation simulations Aim Neonatal cardiopulmonary arrests are rare but serious events. There is limited information on compliance to best-practice guidelines due to rarity, but deviations can have dire consequences. This research aimed to characterize compliance with and deviations from Neonatal Resuscitation Program (NRP) guidelines and their association with teamwork. Methods We observed Emergency Medical Service (EMS) teams responding to standardized neonatal resuscitation simulations following a precipitous home delivery. A Clinical expert evaluated teamwork during simulations using the Clinical Teamwork Scale (CTS™). A neonatologist evaluated technical performance in blinded video review according to NRP guidelines. We report the types, counts, and severity of observed deviations. Logistic regression tested the association of CTS™ factors with the occurrence of deviations. Results Forty-five (45) teams of 265 EMS personnel from fire and transport agencies participated in the simulations. Eighty-seven percent (39/45) of teams were rated as having good teamwork according to CTS™. Nearly all teams (44 of 45) delayed or did not perform one or more of the initial steps of dry, warm, or stimulate; delayed bag-valve mask ventilation (BVM); or performed continuous compressions instead of the recommended 3:1 compression-to-ventilation ratio. Logistic regression revealed an 82% (p < 0.04) decrease in the odds of airway errors for each level of improvement in teams’ decision-making. Conclusion Drying, warming, and stimulating, and ventilation tailored to the physiologic needs of infants continue to be top priorities in neonatal care for out-of-hospital settings. EMS teamwork is good and higher quality of decision-making appears to decrease the odds of ventilation errors. ... [5,9] On the other hand, there is no evidence to prove that the medications used in advanced life support reduce mortality and morbidity, whereas immediate and efficient BLS practices after cardiac arrest are reported to reduce mortality and morbidity. [10] [11][12] People who suffer from sudden cardiac arrest depend on prompt BLS. Patients who receive bystander CPR have a 2-3 time's higher survival rate (8.2% vs. 2.5% for patients who did not receive CPR). [13] Extensive education of the population in particular countries and regions led to high numbers of bystander CPR in cases of out-ofhospital cardiac arrests (OHCA). ... Evaluation of Basic Life Support Knowledge and Skills of Healthcare and Non-healthcare Providers Article Aim: Immediate bystander cardiopulmonary resuscitation (CPR) significantly improves survival after a sudden cardiopulmonary collapse. This study assessed the basic life support (BLS) awareness, knowledge, attitude, and performance of healthcare providers (HCP) and non-HCP before and after CPR training. Materials and Methods: This study included 4625 participants. Participants completed a pre-test to assess their knowledge and 3 h training course that provided a theoretical background on sudden cardiac death and a hands-on CPR tutorial. They were asked to perform BLS on a manikin to simulate an unconscious scenario before the training. Afterward, participants encountered the same scenario and completed a questionnaire of their post-training knowledge. Results: A total of 4625 participants were included in this study. Of which 56.54% (n = 2615) were HCP and 43.45% (n = 2010) were non-HCP. There is a significant increase in knowledge of BLS among non-HCP which is clearly evident in pre-training and post-training evaluation (written and hands-on). Only 0.62% employees are able to perform BLS in the correct sequence before the training and 76.7% employees after the training. None of the students performed BLS in the correct sequence before the training and 60.85% students performed well after the training. Among HCP, only 12.08% were able to perform BLS in the correct sequence before the training and 94.8% after the training. Conclusion: Performing BLS and attending BLS training plays a key role in attaining BLS knowledge by both healthcare and non-HCP ... 19 It has also been shown that after neonatal resuscitation program courses, the number of patients with perinatal asphyxia and also the duration of hospitalization decreased significantly, and the first minute Apgar scores increased significantly. 20 This study is limited by the short duration of data collected as records for the preceding 3 months before the study were not available and at the time of submission, only 6 months data was accessed. It did not also consider sociodemographic factors and intrapartum events such as type and timing of interventions, medications etc and also how exactly the diagnosis of asphyxia as well as its severity was made except for the use Apgar scores. ... Birth Asphyxia among Term Neonates at Korle-Bu Teaching Hospital (KBTH) in Accra ... Delay in performing basic life support could result in a lower probability of a successful result after BLS[4,8]. On the other hand, there is no evidence to prove that the medications used in ALS reduce mortality and morbidity, whereas immediate and efficient BLS practices after cardiac arrest are reported to reduce mortality and morbidity [9] [10][11]. Nurses are the first people to see cases of cardiac arrest in the clinical setting. ... Evaluation of Basic Life Support Training Program Provided for Nurses in A University Hospital Jun 2017 Banu Terzi Şehrinaz Polat Aims: This study was conducted to assess the efficiency of the basic life support (BLS) training program provided for nurses in a university hospital. To evaluate the efficiency of the BLS training program provided for nurses in a university hospital. Methods: In this a quasi-experimental study, a total of 404 nurses who received BLS training were enrolled. The study was performed in two stages. In stage one, the participant nurses were given a pre-test that consisted of 25 questions, four points each, before the training on the first day of the 2-day BLS training. The post-test was conducted in addition to practical exams on manikins to determine nurses’ practice skills on BLS. Results: There was a statistically significant difference between the nurses with previous BLS training and the difference between their pre- and post-test results (p<0.05), and high statistically significant difference was found between the nurses with previous advanced life support (ALS) training and the difference between their pre- and post-test results (p<0.001). Conclusion: Nurses should receive BLS training in hospitals and the training should be repeated on a regular basis. The BLS training that the nurses received in this study was effective and increased their knowledge level on BLS. ... Delay in performing basic life support could result in a lower probability of a successful result after BLS[4,8]. On the other hand, there is no evidence to prove that the medications used in ALS reduce mortality and morbidity, whereas immediate and efficient BLS practices after cardiac arrest are reported to reduce mortality and morbidity [9] [10][11]. Nurses are the first people to see cases of cardiac arrest in the clinical setting. ... Evaluation of Basic Life Support Training Program Provided for Nurses in A University Hospital Aims: This study was conducted to assess the efficiency of the basic life support (BLS) training program provided for nurses in a university hospital. To evaluate the efficiency of the BLS training program provided for nurses in a university hospital. Methods: In this a quasi-experimental study, a total of 404 nurses who received BLS training were enrolled. The study was performed in two stages. In stage one, the participant nurses were given a pre-test that consisted of 25 questions, four points each, before the training on the first day of the 2-day BLS training. The post-test was conducted in addition to practical exams on manikins to determine nurses’ practice skills on BLS. Results: There was a statistically significant difference between the nurses with previous BLS training and the difference between their pre- and post-test results (p ... 3 Neonatal care provided within the first few minutes of life plays a major role in the reduction of neonatal morbidity and mortality. 5 Internationally there is now considerable consensus on how newborn resuscitation should be provided. 6 It is believed that in 95% of cases, when it is required resuscitation should be possible with only a minimum of equipment and without access to intensive care skills or facilities. ... An audit of some health facilities and equipment for neonatal resuscitation in south-south Nigeria Full-text available Jul 2016 Niger J Paediatr Iso Oloyede PA Udo Background: Neonatal Mortality rates continue to be high in spite of the general decline in under-5 mortality rates in Nigeria. Available evidence has shown that the availability of a skilled birth attendant and equipment for basic neonatal resuscitation is necessary for the prevention of early neonatal death which accounts for majority of neonatal mortality. This audit was, therefore, carried out to identify the quantity and quality of equipment available for basic neonatal resuscitation in some health facilities in southern Nigeria.Methods: This was a cross sectional questionnaire based study. Data was obtained from health care workers from 26 health facilities who attended Neonatal resuscitation training workshops from June 2010 to November 2012. Information obtained included type of health facility, obstetric and paediatric services provided and human and material resources available for neonatal resuscitation.Results: Twenty-three (88.5%) were government owned facilities while three (11.5%) were private facilities. Of the government owned facilities four were primary health facilities (17.3%), 18 were secondary (78.4%), while only one (4.3%) was a tertiary health facility. Most of the health facilities had annual deliveries, annual newborn resuscitation and birth asphyxiated babies in the 1-500 range. All the Paediatricians were in the tertiary health facility. Only three (11.5%) facilities had bag and mask and radiant warmer, respectively, while only five (19.2%) had radiant warmers.Conclusion: Equipment for basic neonatal resuscitation is grossly deficient in some health facilities in southern Nigeria. Therefore, urgent efforts should be made by the stakeholders involved in the delivery of child health services to provide basic resuscitation equipment to health facilities. This will contribute to a reduction in the neonatal mortality rate in Nigeria. ... Seemingly, acquiring and maintaining cognitive, technical and behavioural skills is becoming more and more necessary for successfully resuscitating newly born infants (14). Recent studies performed in different environments have showed that implementation of NRP improve not only the skills of the participants, the Apgar scores, shorten the time of hospitalization and improve the quality of care of newly born infants who required resuscitation in the DR (15) . Our study shows that, the majority of the care-givers involved in DR resuscitation is informed of ILCOR recommendations, and has acquired theoretical knowledge and practical skills actively participating in courses organized by the NRG (SEN). ... A survey of neonatal resuscitation in Spain: Gaps between guidelines and practice May 2009 ACTA PAEDIATR Martin Iriondo Marta Thio Lluch E Burón the Neonatal Resuscitation Group (NRG) of the Spanish Neonatal Society (SEN Objectives: To audit the knowledge and application of internationally recommended newborn resuscitation (NR) guidelines among delivery room (DR) caregivers of Spanish hospitals. Methods: A questionnaire-type survey on NR equipment and practices was performed in hospitals of the Spanish National Health System classified according to level of care provided. Results: 88% of the questionnaires were complimented. Limit of viability was set in 23–24 weeks in 78% of the centres. Availability of board-certified and instructors in NRwas significantly higher in level III versus level I–II centres (94 vs. 70% and 78 vs. 51%, respectively). No differences in equipment or knowledge of guidelines of resuscitation were found between centres. Substantial differences were observed in supplementation and monitorization of oxygen, and positive pressure ventilation during resuscitation and transportation. Conclusion: Equipment availability and knowledge of guidelines of NR does not differ between hospitals independent of their level of care. However, performance during resuscitation and transportation in level III hospitals is in significantly greater acquaintance with internationally recommended NR guidelines. Strategic Assessment of Implementing Neonatal Resuscitation Training at a National Level Article Dec 2015 Pediatr Int Lama Charafeddine Maya Badran Pascale Nakad Khalid Yunis Background: Structuring and implementing an endorsed neonatal resuscitation program results in decreased neonatal mortality. This paper aims to evaluate the implementation of formal training using the neonatal resuscitation program (NRP) in a private sector context of a middle income country. Methods: Between 2008 and 2011, the National Collaborative Perinatal Neonatal Network (NCPNN) supported by the Ministry of Health piloted the implementation of NRP training in member hospitals throughout the country. Training of trainers (TOT) then providers (TOP) took place over workshops where a lecture was given followed by hands on training using low fidelity manikins. Pre and post workshop knowledge assessment and hands-on-training evaluation were done. Statistical analysis using SPSS-20 was done with t-test and one way ANOVA to compare mean differences in pre and post scores and percent improvements across specialties and locations. Results: Of 20 TOT participants, 9 (45%) conducted NRP in their hospitals. Ten TOP workshops included 256 professionals. Majority were doctors 128 (50%), 99 (39%) nurses and 20(8%) midwives. Overall pretest scores (67.25%, SD = 16.00) were significantly lower than posttest (87.48%, SD = 11.89) (p = 0.000); percent improvement among all participants was 37.12% (SD = 41.15) (p = 0.82). Nurses and Northern participants had highest mean difference in pre / post scores (21.56; SD = 12.32 and 23.29; SD = 6.62 respectively). Midwives showed the highest percent improvement (Mean = 40.44; SD + 47.28). All but 25 (9.8%) participants passed the "Megacode" from the first attempt. Conclusions: In our setting, implementing NRP training is essential to increase the knowledge and skills of health care professionals. It is sustainable with support from the ministry and other sources. Continuing education should be mandated at the ministry of health level. PO-0814 Developmental Follow Up Of Former Premature Infants: A Pilot Study In Lebanon Article Oct 2014 Lama Charafeddine Racha El Hage Hani Tamim Durriyah D Sinno Background and aims Prematurity is a chronic condition with increased risk of long term disabilities. In middle income countries, paediatricians have to rely on data from western countries since long term follow up of former premature infants are scarce. This pilot study aims at testing the feasibility of a follow up strategy by determining the rate of developmental delay in a sample of former premature infants. Methods This is a cross sectional study of 100 former premature children born at less than 37weeks gestation, from January 2006 to 2009 at a tertiary care centre in Lebanon. Children were evaluated at 3–7 years using Denver Developmental screening test (DDST), Peabody Developmental Motor Scale (PDMS) and Wechsler Preschool and Primary Scale of Intelligence (WPPSI). Analysis was done using SPSS 21 software. Results 86 out of 100 enrolled were tested; their mean age was 4.4 years (+/- 1.5), 56% were males. Conclusion In former preterm infants, the rate of intellectual disability was 14.7% based on WPPSI; 50% had fine motor delay and in total 37% had developmental delay. These data provide objective measures for better distribution of scarce resources in this particular setting. Benefits of simulation based training for neonatal resuscitation education: A systematic review Article Oct 2014 Abhijeet Rakshasbhuvankar Sanjay Patole Background Simulation-based training (SBT) is being more frequently recommended for neonatal resuscitation education (NRE). It is important to assess if SBT improves clinical outcomes as neonatal resuscitation aims to improve survival without long-term neurodevelopmental impairment. We aimed to assess the evidence supporting benefits of SBT in NRE. Method A systematic review was conducted using the Cochrane methodology. PubMed, Embase, PsycInfo and Cochrane databases were searched. Related abstracts were scanned and full texts of the potentially relevant articles were studied. Randomised controlled trials (RCT) and quasi-experimental studies with controls (non-RCT) assessing SBT for NRE were eligible for inclusion in the review. Results Four small studies [three RCT (n = 126) and one non-RCT (n = 60)] evaluated SBT for NRE. Participants included medical students (one RCT and one non-RCT), residents (one RCT) and nursing staff (one RCT). Outcomes included performance in a simulation scenario, theoretical knowledge, and confidence in leading a resuscitation scenario. One RCT favoured simulation [improved resuscitation score (p = 0.016), 2.31 more number of critical actions (p = 0.017) and decreased time to achieve resuscitation steps (p = <0.001)]. The remaining two RCTs and the non-RCT did not find any difference between SBT and alternate methods of instruction. None of the four studies reported clinical outcomes. Conclusions Evidence regarding benefits of SBT for NRE is limited. There are no data on clinical outcomes following SBT for NRE. Large RCTs assessing clinically important outcomes are required before SBT can be recommended widely for NRE. Teaching Medical Students Neonatal Resuscitation: Knowledge Gained and Retained from a Brief Simulation-based Training Workshop Nov 2012 Educ Health Change Learn Pract Nai Ming Lai Chin Fang Ngim Paul Fullerton Context: Despite being an essential clinical skill, many junior doctors feel unprepared to perform neonatal resuscitation. We introduced a neonatal resuscitation training workshop in 2009 for our final-year medical students. Objectives: We assessed the effectiveness of our workshop in improving knowledge immediately post-training and at the end of the year. Methods: We retrospectively analysed the data of our students who attended the workshops during their Paediatric posting in small groups. The workshop was adapted from the American Academy of Paediatrics (AAP) Neonatal Resuscitation Provider (NRP) programme, and included overview lectures, practical simulation, interactive video scenarios and assessments (pre- and post-tests), which comprised 21 multiple-choice questions covering evaluation, practical actions and theory. We repeated the assessment in the final week of the students' medical training ("final test"). We analysed the data using paired t-test, analysis of variance (ANOVA), linear regression and Friedman's test. Findings: All 56 students attended the workshops. Their mean scores (out of 21) were 11.7 (SD 2.5) (pre-test), 16.2 (SD 1.9) (post-test) and 13.6 (SD 2.3) (final test) (P ≤ 0.001 for all pair-wise comparisons). The workshop's timing (earlier or later in the year) had no relationship with the students' final test scores (P = 0.96). In the final test, 68.5%, 67.4% and 60.6% on average answered correctly questions on practical action, theory and evaluation, respectively (P = 0.03). Discussion and conclusions: Our workshop produced a modest gain in student knowledge on neonatal resuscitation at the end of their medical course. The students' overall gain in knowledge was below our expectation, and evaluation appeared to be their weakest domain. Further research should evaluate strategies to enhance longer-term knowledge retention with practical performance. Efficacy and user preference of two CO2 detectors in an infant mannequin randomized crossover trial Full-text available Jun 2013 Eur J Pediatr Gavin Hawkes B J O'Connell Vicki Livingstone Eugene M Dempsey Assessment of effective ventilation in neonatal mask ventilation can be difficult. This study aims to determine whether manual ventilation with a T-piece resuscitator containing an inline CO2 detector (either a Pedi-Cap® CO2 detector or a Neo-StatCO2 <Kg® CO2 detector connected to a facemask) facilitates effective positive pressure ventilation compared to no device in a mannequin study. Paediatric and neonatal trainees were randomly assigned to determine which method they began with (no device, Pedi-Cap or a Neo-Stat). The participants used each method for a period of 3 min. They were video-recorded to determine the amount of effective ventilations delivered and the overall percentage efficiency of each method. Efficacy of ventilation was determined by comparing the number of manual ventilations delivered with the number of times chest rise was observed in the video recording. There were 19 paediatric trainees who provided a total of 7,790 ventilations, and 93% were deemed effective. The percentage of effective ventilations with the T-piece resuscitator alone, the PediCap and the NeoStat were 90, 94 and 96%, respectively. The difference was greatest in the first minute (T-piece resuscitator alone 87.5%, PediCap 94%, NeoStat 96%). Two thirds preferred the Neo-Stat. The use of a CO2 detector improves positive pressure ventilation in a mannequin model, especially in the first minute of positive pressure ventilation. The Neo-Stat CO2 detector was the preferred device by the majority of the participants. Respiratory Distress of the Term Newborn Infant Mar 2013 Martin Oliver Edwards Sarah J. Kotecha Sailesh Kotecha Respiratory distress is recognised as any signs of breathing difficulties in neonates. In the early neonatal period respiratory distress is common, occurring in up to 7% of newborn infants, resulting in significant numbers of term-born infants being admitted to neonatal units. Many risk factors are involved; the increasing number of term infants delivered by elective caesarean section has also increased the incidence. Additionally the risk decreases with each advancing week of gestation. At 37 weeks, the chances are three times greater than at 39-40 weeks gestation. Multiple conditions can present with features of respiratory distress. Common causes in term newborn infants include transient tachypnoea of the newborn, respiratory distress syndrome, pneumonia, meconium aspiration syndrome, persistent pulmonary hypertension of the neonate and pneumothorax. Early recognition of respiratory distress and initiation of appropriate treatment is important to ensure optimal outcomes. This review will discuss these common causes of respiratory distress in term-born infants. What is the impact of structured resuscitation training on healthcare practitioners, their clients and the wider service? A BEME systematic review: BEME Guide No. 20 Jun 2012 MED TEACH Chiara Mosley Chris Dewhurst Stephen Molloy N j Shaw A large number of resuscitation training courses (structured resuscitation training programmes (SRT)) take place in many countries in the world on a regular basis. This review aimed to determine whether after attending SRT programmes, the participants have a sustained retention of resuscitation knowledge and skills after their initial acquisition and whether there is an improvement in outcome for patients and/or their healthcare organisation after the institution of an SRT programme. All research designs were included, and the reported resuscitation training had to have been delivered in a predefined structured manner over a finite period of time. Data was extracted from the 105 eligible articles and research outcomes were assimilated in tabular form with qualitative synthesis of the findings to produce a narrative summary. Findings of the review were: SRTs result in an improvement in knowledge and skills in those who attend them, deterioration in skills and, to a lesser extent, knowledge is highly likely as early as three months following SRTs, booster or refresher sessions may improve an individual's ability to retain resuscitation skills after initial training and the instigation of resuscitation training in a healthcare institution significantly improves clinical management of resuscitations and patient outcome (including survival) after resuscitation attempts. Global report on preterm birth and stillbirth (3 of 7): Evidence for effectiveness of interventions Feb 2010 Fernando C Barros Zulfiqar Bhutta Maneesh Batra Craig Rubens Interventions directed toward mothers before and during pregnancy and childbirth may help reduce preterm births and stillbirths. Survival of preterm newborns may also be improved with interventions given during these times or soon after birth. This comprehensive review assesses existing interventions for low- and middle-income countries (LMICs). Approximately 2,000 intervention studies were systematically evaluated through December 31, 2008. They addressed preterm birth or low birth weight; stillbirth or perinatal mortality; and management of preterm newborns. Out of 82 identified interventions, 49 were relevant to LMICs and had reasonable amounts of evidence, and therefore selected for in-depth reviews. Each was classified and assessed by the quality of available evidence and its potential to treat or prevent preterm birth and stillbirth. Impacts on other maternal, fetal, newborn or child health outcomes were also considered. Assessments were based on an adaptation of the Grades of Recommendation Assessment, Development and Evaluation criteria. Most interventions require additional research to improve the quality of evidence. Others had little evidence of benefit and should be discontinued. The following are supported by moderate- to high-quality evidence and strongly recommended for LMICs: Two interventions prevent preterm births--smoking cessation and progesterone. Eight interventions prevent stillbirths--balanced protein energy supplementation, screening and treatment of syphilis, intermittant presumptive treatment for malaria during pregnancy, insecticide-treated mosquito nets, birth preparedness, emergency obstetric care, cesarean section for breech presentation, and elective induction for post-term delivery. Eleven interventions improve survival of preterm newborns--prophylactic steroids in preterm labor, antibiotics for PROM, vitamin K supplementation at delivery, case management of neonatal sepsis and pneumonia, delayed cord clamping, room air (vs. 100% oxygen) for resuscitation, hospital-based kangaroo mother care, early breastfeeding, thermal care, and surfactant therapy and application of continued distending pressure to the lungs for respiratory distress syndrome The research paradigm for discovery science and intervention development must be balanced to address prevention as well as improve morbidity and mortality in all settings. This review also reveals significant gaps in current knowledge of interventions spanning the continuum of maternal and fetal outcomes, and the critical need to generate further high-quality evidence for promising interventions. Neonatal resuscitation and immediate newborn assessment and stimulation for the prevention of neonatal deaths: A systematic review, meta-analysis and Delphi estimation of mortality effect Apr 2011 BMC PUBLIC HEALTH Anne Cc Lee Simon Cousens Stephen N Wall Joy E Lawn Of 136 million babies born annually, around 10 million require assistance to breathe. Each year 814,000 neonatal deaths result from intrapartum-related events in term babies (previously "birth asphyxia") and 1.03 million from complications of prematurity. No systematic assessment of mortality reduction from tactile stimulation or resuscitation has been published. To estimate the mortality effect of immediate newborn assessment and stimulation, and basic resuscitation on neonatal deaths due to term intrapartum-related events or preterm birth, for facility and home births. We conducted systematic reviews for studies reporting relevant mortality or morbidity outcomes. Evidence was assessed using GRADE criteria adapted to provide a systematic approach to mortality effect estimates for the Lives Saved Tool (LiST). Meta-analysis was performed if appropriate. For interventions with low quality evidence but strong recommendation for implementation, a Delphi panel was convened to estimate effect size. We identified 24 studies of neonatal resuscitation reporting mortality outcomes (20 observational, 2 quasi-experimental, 2 cluster randomized controlled trials), but none of immediate newborn assessment and stimulation alone. A meta-analysis of three facility-based studies examined the effect of resuscitation training on intrapartum-related neonatal deaths (RR= 0.70, 95%CI 0.59-0.84); this estimate was used for the effect of facility-based basic neonatal resuscitation (additional to stimulation). The evidence for preterm mortality effect was low quality and thus expert opinion was sought. In community-based studies, resuscitation training was part of packages with multiple concurrent interventions, and/or studies did not distinguish term intrapartum-related from preterm deaths, hence no meta-analysis was conducted. Our Delphi panel of 18 experts estimated that immediate newborn assessment and stimulation would reduce both intrapartum-related and preterm deaths by 10%, facility-based resuscitation would prevent a further 10% of preterm deaths, and community-based resuscitation would prevent further 20% of intrapartum-related and 5% of preterm deaths. Neonatal resuscitation training in facilities reduces term intrapartum-related deaths by 30%. Yet, coverage of this intervention remains low in countries where most neonatal deaths occur and is a missed opportunity to save lives. Expert opinion supports smaller effects of neonatal resuscitation on preterm mortality in facilities and of basic resuscitation and newborn assessment and stimulation at community level. Further evaluation is required for impact, cost and implementation strategies in various contexts. This work was supported by the Bill & Melinda Gates Foundation through a grant to the US Fund for UNICEF, and to the Saving Newborn Lives program of Save the Children, through Save the Children US.	https://www.researchgate.net/publication/222910096_The_impact_of_Neonatal_Resuscitation_Program_courses_on_mortality_and_morbidity_of_newborn_infants_with_perinatal_asphyxia
The Characterization of Postoperative Mechanical Respiratory Requirement in Neonates and Infants Undergoing Cardiac Surgery on Cardiopulmonary Bypass in a Single Tertiary Institution \| Request PDF Request PDF \| The Characterization of Postoperative Mechanical Respiratory Requirement in Neonates and Infants Undergoing Cardiac Surgery on Cardiopulmonary Bypass in a Single Tertiary Institution \| Objectives Although neonates and infants undergoing cardiac surgery on cardiopulmonary bypass (CPB) are at high risk of developing perioperative... \| Find, read and cite all the research you need on ResearchGate The Characterization of Postoperative Mechanical Respiratory Requirement in Neonates and Infants Undergoing Cardiac Surgery on Cardiopulmonary Bypass in a Single Tertiary Institution April 2021 Journal of Cardiothoracic and Vascular Anesthesia 36(1) DOI: 10.1053/j.jvca.2021.04.023 Authors: Sheng Xiang Huang Karina Lukovits Abstract Objectives Although neonates and infants undergoing cardiac surgery on cardiopulmonary bypass (CPB) are at high risk of developing perioperative morbidity and mortality, including lung injury, the intraoperative profile of lung injury in this cohort is not well-described. Given that the postoperative course of patients in the pediatric cardiac surgical arena has become increasingly expedited, the objective of this study was to characterize the profiles of postoperative mechanical ventilatory support in neonates and infants undergoing cardiac surgery on CPB and to examine the characteristics of lung mechanics and lung injury in this patient population who are potentially amendable to early postoperative recovery in a single tertiary pediatric institution. Design A retrospective data analysis of neonates and infants who underwent cardiac surgery on cardiopulmonary bypass. Setting A single-center, university teaching hospital. Participants The study included 328 neonates and infants who underwent cardiac surgery on cardiopulmonary bypass. Interventions A subset of 128 patients were studied: 58 patients undergoing ventricular septal defect (VSD) repair, 36 patients undergoing complete atrioventricular canal (CAVC) repair, and 34 patients undergoing bidirectional Glenn (BDG) shunt surgery. Measurements and Main Results Of the entire cohort, 3.7% experienced in-hospital mortality. Among all surgical procedures, VSD repair (17.7%) was the most common, followed by CAVC repair (11.0%) and BDG shunt surgery (10.4%). Of patients who underwent VSD repair, CAVC repair, and BDG shunt surgery, 65.5%, 41.7%, and 67.6% were off mechanical ventilatory support within 24 hours postoperatively, respectively. In all three of the surgical repairs, lung compliance decreased after CPB compared to pre-CPB phase. Sixty point three percent of patients with VSD repair and 77.8% of patients with CAVC repair showed a PaO2/FIO2 (P/F) ratio of <300 after CPB. Post- CPB P/F ratios of 120 for VSD patients and 100 for CAVC patients were considered as optimal cutoff values to highly predict prolonged (>24 hours) postoperative mechanical ventilatory support. A higher volume of transfused platelets also was associated with postoperative ventilatory support ≥24 hours in patients undergoing VSD repair, CAVC repair, and BDG shunt surgery. Conclusions There was a high incidence of lung injury after CPB in neonates and infants, even in surgeries amendable for early recovery. Given that CPB-related factors (CPB duration, crossclamp time) and volume of transfused platelet were significantly associated with prolonged postoperative ventilatory support, the underlying cause of cardiac surgery-related lung injury can be multi-factorial. Effect of Intraoperative Dexamethasone on Major Complications and Mortality Among Infants Undergoing Cardiac Surgery: The DECISION Randomized Clinical Trial Jun 2020 Vladimir Lomivorotov Igor Kornilov Vladimir A Boboshko Dmitry N Ponomarev Importance Corticosteroids are widely used in pediatric cardiac surgery to blunt systemic inflammatory response and to reduce complications; nevertheless, their clinical efficacy is uncertain. Objective To determine whether intraoperative administration of dexamethasone is more effective than placebo for reducing major complications and mortality during pediatric cardiac surgery. Design, Setting, and Participants The Intraoperative Dexamethasone in Pediatric Cardiac Surgery was an investigator-initiated, double-blind, multicenter randomized trial that involved 4 centers in China, Brazil, and Russia. A total of 394 infants younger than 12 months, undergoing cardiac surgery with cardiopulmonary bypass were enrolled from December 2015 to October 2018, with follow-up completed in November 2018. Interventions The dexamethasone group (n = 194) received 1 mg/kg of dexamethasone; the control group (n = 200) received an equivolume of 0.9% sodium chloride intravenously after anesthesia induction. Main Outcomes and Measures The primary end point was a composite of death, nonfatal myocardial infarction, need for extracorporeal membrane oxygenation, need for cardiopulmonary resuscitation, acute kidney injury, prolonged mechanical ventilation, or neurological complications within 30 days after surgery. There were 17 secondary end points, including the individual components of the primary end point, and duration of mechanical ventilation, inotropic index, intensive care unit stay, readmission to intensive care unit, and length of hospitalization. Results All of the 394 patients randomized (median age, 6 months; 47.2% boys) completed the trial. The primary end point occurred in 74 patients (38.1%) in the dexamethasone group vs 91 patients (45.5%) in the control group (absolute risk reduction, 7.4%; 95% CI, −0.8% to 15.3%; hazard ratio, 0.82; 95% CI, 0.60 to 1.10; P = .20). Of the 17 prespecified secondary end points, none showed a statistically significant difference between groups. Infections occurred in 4 patients (2.0%) in the dexamethasone group vs 3 patients (1.5%) in the control group. Conclusions and Relevance Among infants younger than 12 months undergoing cardiac surgery with cardiopulmonary bypass, intraoperative administration of dexamethasone, compared with placebo, did not significantly reduce major complications and mortality at 30 days. However, the study may have been underpowered to detect a clinically important difference. Trial Registration ClinicalTrials.gov Identifier: NCT02615262 Volatile Anesthetics, Not Intravenous Anesthetic Propofol Bind to and Attenuate the Activation of Platelet Receptor Integrin αIIbβ3 PLOS ONE Koichi Yuki Weiming Bu Motomu Shimaoka Roderic G. Eckenhoff In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3. The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis. Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation. Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner. Sedative Drug Modulates T-Cell and Lymphocyte Function-Associated Antigen-1 Function Mar 2011 Koichi Yuki Sulpicio G Soriano Motomu Shimaoka Sedative drugs modify immune cell functions via several mechanisms. However, the effects of sedatives on immune function have been primarily investigated in neutrophils and macrophages, and to the lesser extent lymphocytes. Lymphocyte function-associated antigen-1 (LFA-1) is an adhesion molecule that has a central role in regulating immune function of lymphocytes including interleukin-2 (IL-2) production and lymphocyte proliferation. Previous clinical studies reported that propofol and isoflurane reduced IL-2 level in patients, but midazolam did not. We previously demonstrated that isoflurane inhibited LFA-1 binding to its counter ligand, intercellular adhesion molecule-1 (ICAM-1), which might contribute to the reduction of IL-2 levels. In the current study, we examined the effect of propofol, midazolam, and dexmedetomidine on LFA-1/ICAM-1 binding, and the subsequent biological effects. The effect of sedative drugs on T-cell proliferation and IL-2 production was measured by calorimetric assays on human peripheral blood mononuclear cells. Because LFA-1/ICAM-1 binding has an important role in T-cell proliferation and IL-2 production, we measured the effect of sedative drugs on ICAM-1 binding to LFA-1 protein (cell-free assay). This analysis was followed by flow cytometric analysis of LFA-1 expressing T-cell binding to ICAM-1 (cell-based assay). To determine whether the drug/LFA-1 interaction is caused by competitive or allosteric inhibition, we analyzed the sedative drug effect on wild-type and high-affinity LFA-1 and a panel of monoclonal antibodies that bind to different regions of LFA-1. Propofol at 10 to 100 μM inhibited ICAM-1 binding to LFA-1 in cell-free assays and cell-based assays (P < 0.05). However, dexmedetomidine and midazolam did not affect LFA-1/ICAM-1 binding. Propofol directly inhibits LFA-1 binding to ICAM-1 by binding near the ICAM-1 contact area in a competitive manner. At clinically relevant concentrations, propofol, but not dexmedetomidine or midazolam, inhibited IL-2 production (P < 0.05). Additionally, propofol inhibited lymphocyte proliferation (P < 0.05). Our study suggests that propofol competitively inhibits LFA-1 binding to ICAM-1 on T-cells and suppresses T-cell proliferation and IL-2 production, whereas dexmedetomidine and midazolam do not significantly influence these immunological assays. Mortality Associated With Congenital Heart Defects in the United States: Trends and Racial Disparities, 1979–1997 Jun 2001 CIRCULATION Roumiana Boneva Lorenzo Botto Cynthia Moore J. David Erickson Surgical series and some population-based studies have documented a decrease in mortality from heart defects. Recent population-based data for the United States are lacking, however. We examined population-based data for patterns, time trends, and racial differences of mortality from heart defects for the United States from 1979 through 1997. We examined the multiple-cause mortality files compiled by the National Center for Health Statistics of the CDC from all death certificates filed in the United STATES: From these data, we derived death rates (deaths per 100 000 population) by the decedent's age, race, year of death, and heart defect type. We also analyzed age at death as an indirect indicator of survival. From 1979 through 1997, mortality from heart defects (all ages) declined 39%, from 2.5 to 1.5 per 100 000 population; among infants, the decline was 39%, or 2.7% per year. In 1995 to 1997, heart defects contributed to 5822 deaths per year. Of these deaths, 51% were among infants and 7% among children 1 to 4 years old. Mortality was on average 19% higher among blacks than among whites; this gap does not appear to be closing. Age at death increased for most heart defects, although less among blacks than among whites. Mortality from heart defects is declining in the United States, although it remains a major cause of death in infancy and childhood. Age at death is increasing, suggesting that more affected persons are living to adolescence and adulthood. The racial discrepancies should be investigated to identify opportunities for prevention. Oct 2019 J THORAC CARDIOV SUR Nathalie Roy Maria Fernanda Parra Morgan L Brown Pedro del Nido Objective: We hypothesized that a new enhanced recovery after surgery (ERAS) program would accelerate functional recovery after congenital heart surgery and reduce length of stay and complications. Methods: Evidence-based interventions in perioperative care were evaluated for relevance, and components of the ERAS cardiac program were determined. The target patient population included infants to adults with low comorbidities. Major outcomes were compared to a pre-ERAS era cohort using propensity matching. Results: From October 1, 2018, to February 28, 2019, 155 of 448 patients were eligible for the ERAS program. The median age was 3.6 years (interquartile range, 0.5-12.3). Key metrics included early extubation (<8 hours), achieved in 84 patients (54%; median 7.6 hours; interquartile range, 3.8-12.3), and multimodal pain regimen used in all patients (100%) postoperatively, but in only 88 of 155 patients (57%) intraoperatively. Opioid analgesia was highest the night of surgery (oral morphine equivalent: 0.36 mg/kg/12 hours; interquartile range, 0.21-0.57). In matched analysis, raw median mechanical ventilation time was 7.6 hours (interquartile range, 3.8-12.2) in ERAS versus 8.2 (interquartile range, 4.0-17.0) in pre-ERAS era (P = .001 log-hours). Raw median intensive care unit length of stay was shorter with ERAS: 1.12 days (interquartile range, 0.93-2.01) versus 1.28 days (interquartile range, 0.96-2.09) pre-ERAS (P = .046 log-days), but there was no difference in hospital length of stay. There was no increase in Society of Thoracic Surgeons-reported complications, readmissions, and reinterventions. Conclusions: This represents the initial implementation experience of an enhanced recovery after surgery program after congenital surgery at a large pediatric hospital. Adherence to the program component metrics is not yet optimized, but monthly sharing of quality metrics allows multidisciplinary collaboration, provider engagement, and opportunities for research and process improvement. Acute respiratory distress syndrome Mar 2019 Michael A Matthay Rachel L Zemans Guy A. Zimmerman Carolyn S Calfee The acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in critically ill patients and is defined by the acute onset of noncardiogenic pulmonary oedema, hypoxaemia and the need for mechanical ventilation. ARDS occurs most often in the setting of pneumonia, sepsis, aspiration of gastric contents or severe trauma and is present in ~10% of all patients in intensive care units worldwide. Despite some improvements, mortality remains high at 30-40% in most studies. Pathological specimens from patients with ARDS frequently reveal diffuse alveolar damage, and laboratory studies have demonstrated both alveolar epithelial and lung endothelial injury, resulting in accumulation of protein-rich inflammatory oedematous fluid in the alveolar space. Diagnosis is based on consensus syndromic criteria, with modifications for under-resourced settings and in paediatric patients. Treatment focuses on lung-protective ventilation; no specific pharmacotherapies have been identified. Long-term outcomes of patients with ARDS are increasingly recognized as important research targets, as many patients survive ARDS only to have ongoing functional and/or psychological sequelae. Future directions include efforts to facilitate earlier recognition of ARDS, identifying responsive subsets of patients and ongoing efforts to understand fundamental mechanisms of lung injury to design specific treatments. The effects of positive airway pressure and mechanical ventilation of the lungs during cardiopulmonary bypass on pulmonary adverse events after cardiac surgery: A systematic review and meta-analysis Article Sep 2017 J CARDIOTHOR VASC AN Yichia Wang Chi-Hsiang Huang Yu-Kang Tu Objective To investigate whether different ventilation strategies during cardiopulmonary bypass (CPB) can improve the outcomes in adult cardiac surgery patients. Design Systematic review of randomized controlled trials with meta-analyses. Setting Clinical trials for human studies with no high-risk bias up to July 2016 were obtained from electronic databases (Medline, EMBASE, PubMed, and the Cochrane Central Register of Controlled Trials, and reference lists of relevant randomized trials and review articles.) Participants adult patients undergoing cardiac surgery Interventions patients receiving cardiac surgery with CPB and ventilation or continuous positive airway pressure (CPAP) Measurements and Main Results Fifteen randomized controlled trials with 748 patients were analyzed. In cardiac surgery, CPAP use during CPB was associated with an improved A–a gradient difference compared with no CPAP (weighted mean difference [WMD] = 4.11 kPa; 95% confidence interval [CI] = 0.85–7.37; I-squared value = 28.8%). Ventilation during CPB did not improve the postoperative hypoxemia score (WMD = 30.94; 95% CI, −20.76 to 82.63; I-squared = 61%) or diffusion capacity compared with the apnea group (WMD = 2.59 kPa; 95% CI = -2.49–7.67; I-squared value= 81.3%). Neither CPAP nor ventilation during CPB was associated with a shorter mechanical ventilation time or hospital stay. Conclusions CPAP during CPB improved A–a gradient difference compared with apnea, but ventilation during CPB did not. Neither CPAP nor ventilation during CPB had evidence to improve clinical outcomes in low- or intermediate-risk patients for elective cardiac surgery. The findings are inconclusive because of heterogeneity and small sample sizes. Adaptation and Validation of a Pediatric Sequential Organ Failure Assessment Score and Evaluation of the Sepsis-3 Definitions in Critically Ill Children Aug 2017 Travis Matics L. Nelson Sanchez-Pinto Importance The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) uses the Sequential Organ Failure Assessment (SOFA) score to grade organ dysfunction in adult patients with suspected infection. However, the SOFA score is not adjusted for age and therefore not suitable for children. Objectives To adapt and validate a pediatric version of the SOFA score (pSOFA) in critically ill children and to evaluate the Sepsis-3 definitions in patients with confirmed or suspected infection. Design, Setting, and Participants This retrospective observational cohort study included all critically ill children 21 years or younger admitted to a 20-bed, multidisciplinary, tertiary pediatric intensive care unit between January 1, 2009 and August 1, 2016. Data on these children were obtained from an electronic health record database. The pSOFA score was developed by adapting the original SOFA score with age-adjusted cutoffs for the cardiovascular and renal systems and by expanding the respiratory criteria to include noninvasive surrogates of lung injury. Daily pSOFA scores were calculated from admission until day 28 of hospitalization, discharge, or death (whichever came first). Three additional pediatric organ dysfunction scores were calculated for comparison. Exposures Organ dysfunction measured by the pSOFA score, and sepsis and septic shock according to the Sepsis-3 definitions. Main Outcomes and Measures The primary outcome was in-hospital mortality. The daily pSOFA scores and additional pediatric organ dysfunction scores were compared. Performance was evaluated using the area under the curve. The pSOFA score was then used to assess the Sepsis-3 definitions in the subgroup of children with confirmed or suspected infection. Results In all, 6303 patients with 8711 encounters met inclusion criteria. Each encounter was treated independently. Of the 8482 survivors of hospital encounters, 4644 (54.7%) were male and the median (interquartile range [IQR]) age was 69 (17-156) months. Among the 229 nonsurvivors, 127 (55.4%) were male with a median (IQR) age of 43 (8-144) months. In-hospital mortality was 2.6%. The maximum pSOFA score had excellent discrimination for in-hospital mortality, with an area under the curve of 0.94 (95% CI, 0.92-0.95). The pSOFA score had a similar or better performance than other pediatric organ dysfunction scores. According to the Sepsis-3 definitions, 1231 patients (14.1%) were classified as having sepsis and had a mortality rate of 12.1%, and 347 (4.0%) had septic shock and a mortality rate of 32.3%. Patients with sepsis were more likely to die than patients with confirmed or suspected infection but no sepsis (odds ratio, 18; 95% CI, 11-28). Of the 229 patients who died during their hospitalization, 149 (65.0%) had sepsis or septic shock during their course. Conclusions and Relevance The pSOFA score was adapted and validated with age-adjusted variables in critically ill children. Using the pSOFA score, the Sepsis-3 definitions were assessed in children with confirmed or suspected infection. This study is the first assessment, to date, of the Sepsis-3 definitions in critically ill children. Use of these definitions in children is feasible and shows promising results. Monitoring of lung function in acute respiratory distress syndrome Jul 2017 Anders Larsson Claude Guerin Monitoring of lung function is essential to assess changes in the lung condition, and to correct and improve ventilator and adjuvant therapies in acute respiratory distress syndrome (ARDS). In this review we discuss the use of monitoring of gas exchange, lung mechanics and shortly on lung imaging in this condition. View Show abstract Prolonged exposure to volatile anesthetic isoflurane worsens the outcome of polymicrobial abdominal sepsis Dec 2016 TOXICOL SCI Sophia Koutsogiannaki Matthew M. Schaefers Toshiaki Okuno Koichi Yuki Sepsis continues to result in high morbidity and mortality. General anesthesia is often administered to septic patients, but the impacts of general anesthesia on host defense are not well understood. General anesthesia can be given by volatile and intravenous anesthetics. Our previous in vitro study showed that volatile anesthetic isoflurane directly inhibits leukocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1), critical adhesion molecules on leukocytes. Thus, the role of isoflurane exposure on in vivo LFA-1 and Mac-1 function was examined using polymicrobial abdominal sepsis model in mice. As a comparison, intravenous anesthetic propofol was given to a group of mice. Wild type, LFA-1, Mac-1 and ICAM-1 knockout mice were used. Following the induction of polymicrobial abdominal sepsis by cecal ligation and puncture, groups of mice were exposed to isoflurane for either 2 or 6 hours, or to propofol for 6 hours, and their outcomes were examined. Bacterial loads in tissues and blood, neutrophil recruitment to the peritoneal cavity and phagocytosis were studied. 6 hours of isoflurane exposure worsened the outcome of abdominal sepsis (p< 0.0001) with higher bacterial loads in tissues, but 2 hours of isoflurane or 6 hours of propofol exposure did not. Isoflurane impaired neutrophil recruitment to the abdominal cavity by inhibiting LFA-1 function. Isoflurane also impaired bacterial phagocytosis via complement receptors including Mac-1. In conclusion, prolonged isoflurane exposure worsened the outcome of experimental polymicrobial abdominal sepsis and was associated with impaired neutrophil recruitment and bacterial phagocytosis via reduced LFA-1 and Mac-1 function. The effect of different anesthetics on tumor cytotoxicity by natural killer cells Dec 2016 Kazumasa Tazawa Sophia Koutsogiannaki Matthew Chamberlain Koichi Yuki A number of retrospective studies have suggested that choice of anesthetic drugs during surgical tumor resection might affect tumor recurrence/metastasis, or outcome of patients. The recent study showed that volatile anesthetics-based general anesthesia was associated with the worse outcomes than intravenous anesthetics-based general anesthesia. However, the underlying mechanism is yet to be determined. Because natural killer (NK) cells are implicated as important immune cells for tumor recurrence/metastasis in the perioperative period, we examined the effect of different anesthetics on NK cell-mediated tumor cytotoxicity. Because adhesion molecule leukocyte function-associated antigen-1 (LFA-1) is functionally important in NK cells and is inhibited by commonly used volatile anesthetics isoflurane and sevoflurane, we hypothesized that these anesthetics would attenuate NK cell-mediated cytotoxicity. Using human NK cell line NK92-MI cells and tumor cell line K562 cells as a model system, we performed cytotoxicity, proliferation, conjugation and degranulation assays. Lytic granule polarization was also assessed. We showed that isoflurane, sevoflurane and LFA-1 inhibitor BIRT377 attenuated cytotoxicity, and reduced conjugation and polarization, but not degranulation of NK cells. Our data suggest that isoflurane and sevoflurane attenuated NK cell-mediated cytotoxicity at least partly by their LFA-1 inhibition in vitro. Whether or not isoflurane and sevoflurane attenuate NK cell-mediated tumor cytotoxicity in patients needs to be determined in the future. Cardiopulmonary Bypass in the Pediatric Population Article Apr 2015 Best Pract Res Clin Anaesthesiol David Whiting Koichi Yuki James Dinardo Cardiopulmonary bypass (CPB) facilitates the repair of cardiac lesions in adults and children. Surgical mortality has decreased with improvements in technique allowing for the successful repair of complex heart defects in neonates of increasingly low body weight and gestational age. The physiological effects of CPB are more significant in children. The presence of intracardiac shunts and other anatomic variants further complicates CPB in patients with congenital heart disease. Special techniques and monitors are often necessary. Protocols are often established within individual institutions to standardize the approach to CPB. The anesthesiologist caring for the patient must understand the physiology of CPB to facilitate the initiation and separation from bypass, and to be able to treat complications. Evidence supporting a particular technique of CPB in pediatric population is still largely from uncontrolled or nonrandomized trials, observational studies, extrapolation from adult studies, and expert opinion. The heterogeneity of congenital heart disease makes randomized controlled trials or meta-analyses challenging, and thus they are limited in the literature. Copyright © 2015 Elsevier Ltd. All rights reserved. Should early extubation be the goal for children after congenital cardiac surgery? Jul 2014 J THORAC CARDIOV SUR Kevin C Harris Spencer Holowachuk Sandy Pitfield Sanjiv K Gandhi Objective: We sought to determine the feasibility and assess the clinical outcomes associated with an early extubation strategy for all children undergoing congenital heart surgery, including neonates (age, <30 days). Methods: We performed a linked database analysis of all patients undergoing congenital heart surgery from July 1, 2010 to December 31, 2012. We collected data on the cardiac diagnoses, preoperative status, procedure, and postoperative course, including the duration of invasive and noninvasive ventilation, failure of extubation, hemodynamic data, length of stay, complications, and mortality. A multivariable model was used to assess the independent factors associated with an inability to extubate within the operating room and with delayed extubation (>24 hours). Results: We operated on 613 children, including 97 neonates. Intraoperative extubation was achieved in 71% of the cases and early extubation (≤ 24 hours) was achieved in 89% of the cases. The overall mortality was 1.5% (9 of 613 patients). Early extubation was associated with lower mortality (1% vs 9%, P < .001) and a lower rate of reintubation (4% vs 23%, P < .001) compared with delayed extubation. Notably, 63% of the neonates were extubated within 24 hours, including 67% of arterial switch operations and 54% of total anomalous pulmonary venous return repairs. Norwood operations were the only procedure in which no patient was extubated within the first 24 hours. Multivariable logistic regression demonstrated that the predictors of delayed extubation included preoperative mechanical ventilation, weight < 5 kg, a longer procedure time, and the need for postoperative inotrope support. Implementation of an early extubation strategy was associated with low rates of complications (5.1 per 10 procedures), short lengths of intensive care unit stay (median, 1 day; interquartile range, 1-3), and short hospital stays (median, 4 days; interquartile range, 3-6). Conclusions: Most children undergoing congenital heart surgery can be extubated in the operating room. Most neonates, including many undergoing complex procedures, can be extubated within the first 24 hours after surgery. Early extubation was associated with low morbidity rates and short lengths of intensive care unit and hospital stays. Propofol Shares the Binding Site with Isoflurane and Sevoflurane on Leukocyte Function-Associated Antigen-1 Aug 2013 Koichi Yuki Weiming Bu Jin Xi Roderic G. Eckenhoff Background: We previously demonstrated that propofol interacted with the leukocyte adhesion molecule leukocyte function-associated antigen-1 (LFA-1) and inhibited the production of interleukin-2 via LFA-1 in a dependent manner. However, the binding site(s) of propofol on LFA-1 remains unknown. Methods: First, the inhibition of LFA-1's ligand binding by propofol was confirmed in an enzyme-linked immunosorbent assay (ELISA) ELISA-type assay. The binding site of propofol on LFA-1 was probed with a photolabeling experiment using a photoactivatable propofol analog called azi-propofol-m. The adducted residues of LFA-1 by this compound were determined using liquid chromatography-mass spectrometry. In addition, the binding of propofol to the ligand-binding domain of LFA-1 was examined using 1-aminoanthracene (1-AMA) displacement assay. Furthermore, the binding site(s) of 1-AMA and propofol on LFA-1 was studied using the docking program GLIDE. Results: We demonstrated that propofol impaired the binding of LFA-1 to its ligand intercellular adhesion molecule-1. The photolabeling experiment demonstrated that the adducted residues were localized in the allosteric cavity of the ligand-binding domain of LFA-1 called "lovastatin site." The shift of fluorescence spectra was observed when 1-AMA was coincubated with the low-affinity conformer of LFA-1 ligand-binding domain (wild-type [WT] αL I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT αL I domain. In the 1-AMA displacement assay, propofol decreased 1-AMA fluorescence signal (at 520 nm), suggesting that propofol competed with 1-AMA and bound to the WT αL I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment. Conclusions: We demonstrated that propofol bound to the lovastatin site in LFA-1. Previously we showed that the volatile anesthetics isoflurane and sevoflurane bound to this site. Taken together, the lovastatin site is an example of the common binding sites for anesthetics currently used clinically. Isoflurane inhibits neutrophil recruitment in the cutaneous Arthus reaction model Oct 2012 J ANESTH Carla Carbo Koichi Yuki Melanie Demers Motomu Shimaoka Purpose: Neutrophil recruitment to the inflammatory sites is regulated by a variety of adhesion molecules including β2 integrins. The dependency of neutrophil recruitment on β2 integrins is variable in different tissues, but has not yet been verified in the cutaneous passive reverse Arthus reaction. We examined this question and also evaluated the impact of isoflurane on neutrophil recruitment to the skin because we previously showed in vitro that isoflurane binds and inhibits β2 integrins. Methods: The dependency on β2 integrins in neutrophil recruitment to the skin in the Arthus reaction was examined using αL, αM and β2 knockout mice. Then, we evaluated the effect of isoflurane on neutrophil recruitment to the skin. In addition, the effects of isoflurane on neutrophil binding to intercellular adhesion molecule-1 (ICAM-1), one of the β2 integrin ligands, were studied in vitro using cell adhesion assays. Results: Neutrophil recruitment to the skin in the Arthus reaction model was totally dependent on β2 integrins, as β2 knockout mice completely abolished it. However, the defect of only one of the β2 integrins was not sufficient to abolish neutrophil recruitment. Isoflurane reduced neutrophil recruitment to the skin by approximately 90 %. Also, isoflurane inhibited neutrophil adhesion to β2 integrin ligand ICAM-1. Conclusions: We demonstrated that (1) neutrophil recruitment to the skin was totally dependent on β2 integrins, and (2) isoflurane significantly impaired neutrophil recruitment. Based on the previous studies on the contribution of other adhesion molecules in neutrophil recruitment, it is likely that isoflurane at least partially affects on β2 integrins in this model. Anesthetic management of noncardiac surgery for patients with single ventricle physiology Apr 2011 J ANESTH Koichi Yuki Alfonso Casta Shoichi Uezono Patients with congenital heart diseases are a growing population, and noncardiac surgeries will become an important health care issue. Patients with single ventricle physiology are a particularly challenging population who will undergo staged, palliative repair toward a final step of Fontan circulation. Although Fontan surgery creates a serial circulation in which the ventricle pumps blood to the systemic circuit, pulmonary blood flow occurs without a dedicated ventricle. Despite progress in outcomes, this abnormal circulation remains associated with various co-morbidities such as ventricular dysfunction, arrhythmias, protein losing enteropathy, and plastic bronchitis. Health care professionals must prepare for these patients to present to noncardiac surgery at any stage of intervention, possibly with complications. Given that staged, palliative repair has undergone multiple modifications, patients who present for surgery can vary in types and timing of the repair. Anesthesiologists who care for them must be familiar with perioperative issues to optimize outcomes, especially because congenital heart disease is a risk factor for increased mortality for noncardiac surgery. Mortality Resulting From Congenital Heart Disease Among Children and Adults in the United States, 1999 to 2006 Nov 2010 CIRCULATION Suzanne M Gilboa Jason Lee Salemi Wendy Nembhard Adolfo Correa Previous reports suggest that mortality resulting from congenital heart disease (CHD) among infants and young children has been decreasing. There is little population-based information on CHD mortality trends and patterns among older children and adults. We used data from death certificates filed in the United States from 1999 to 2006 to calculate annual CHD mortality by age at death, race-ethnicity, and sex. To calculate mortality rates for individuals ≥1 year of age, population counts from the US Census were used in the denominator; for infant mortality, live birth counts were used. From 1999 to 2006, there were 41,494 CHD-related deaths and 27,960 deaths resulting from CHD (age-standardized mortality rates, 1.78 and 1.20 per 100,000, respectively). During this period, mortality resulting from CHD declined 24.1% overall. Mortality resulting from CHD significantly declined among all race-ethnicities studied. However, disparities persisted; overall and among infants, mortality resulting from CHD was consistently higher among non-Hispanic blacks compared with non-Hispanic whites. Infant mortality accounted for 48.1% of all mortality resulting from CHD; among those who survived the first year of life, 76.1% of deaths occurred during adulthood (≥18 years of age). CHD mortality continued to decline among both children and adults; however, differences between race-ethnicities persisted. A large proportion of CHD-related mortality occurred during infancy, although significant CHD mortality occurred during adulthood, indicating the need for adult CHD specialty management. Sex Differences in Mortality in Children Undergoing Congenital Heart Disease Surgery A United States Population-Based Study Sep 2010 CIRCULATION Ariane Marelli Kimberlee Gauvreau Mike Landzberg Kathy Jenkins The changing demographics of the adult congenital heart disease (CHD) population requires an understanding of the factors that impact patient survival to adulthood. We sought to investigate sex differences in CHD surgical mortality in children. Children <18 years old hospitalized for CHD surgery were identified using the Kids' Inpatient Database in 2000, 2003, and 2006. Demographic, diagnostic, and procedural variables were grouped according to RACHS-1 (Risk Adjustment for Congenital Heart Surgery) method. Logistic regression was used to determine the odds ratio of death in females versus males adjusting for RACHS-1 risk category, age, prematurity, major noncardiac anomalies, and multiple procedures. Analyses were stratified by RACHS-1 risk categories and age. Of 33 848 hospitalizations for CHD surgery, 54.7% were in males. Males were more likely than females to have CHD surgery in infancy, high-risk CHD surgery, and multiple CHD procedures. Females had more major noncardiac structural anomalies and more low-risk procedures. However, the adjusted risk of in-hospital death was higher in females (odds ratio, 1.21; 95% confidence interval, 1.08 to 1.36) on account of the subgroup with high-risk surgeries who were <1 year of age (odds ratio, 1.39; 95% confidence interval, 1.16 to 1.67). In this large US population study, more male children underwent CHD surgery and had high-risk procedures. Female infants who had high-risk procedures were at higher risk for death, but this accounted for a small proportion of females and is therefore unlikely to have a major impact on the changing demographics in adults in CHD. Pulmonary injury after cardiopulmonary bypass: Beneficial effects of low-frequency mechanical ventilation Jul 2009 Hajime Imura Massimo Caputo Kelvin H H Lim Gianni Angelini Pulmonary dysfunction is a frequent postoperative complication after cardiac surgery with cardiopulmonary bypass, and atelectasis is thought to be one of the main causes. The aim of this study was to evaluate whether low-frequency ventilation and continuous positive airway pressure during cardiopulmonary bypass reduce postcardiopulmonary bypass lung injury. Eighteen Yorkshire pigs were subjected to 120 minutes of cardiopulmonary bypass (1 hour of cardioplegic arrest) followed by 90 minutes of recovery before being sacrificed. Six animals served as control with the endotracheal tube open to atmosphere during cardiopulmonary bypass. The remaining animals were divided into 2 groups of 6: One group received continuous positive airway pressure of 5 cm H(2)O, and one group received low-frequency ventilation (5/minutes) during cardiopulmonary bypass. Lung tissue biopsy and bronchoalveolar lavage samples were obtained before and 90 minutes after discontinuation of cardiopulmonary bypass for measurement of adenine nucleotide (adenosine-5'-triphosphate, adenosine diphosphate, adenosine monophosphate), lactate dehydrogenase, DNA levels, and histology. Hemodynamic data and arterial blood gases were also collected through the study. The hemodynamic parameters were similar in the 3 groups. After cardiopulmonary bypass, the low-frequency ventilation group showed significantly better oxygen tension and alveolar arterial oxygen gradient, higher adenine nucleotide, lower lactate dehydrogenase levels, and reduced histologic damage in lung biopsy, as well as lower DNA levels in bronchoalveolar lavage compared with the control group. The continuous positive airway pressure group showed only significantly reduced lactate dehydrogenase levels compared with control. Low-frequency ventilation during cardiopulmonary bypass in a pig experimental model reduces tissue metabolic and histologic damage in the lungs and is associated with improved postoperative gas exchange. Congenital Heart Disease in 56,109 Births Incidence and Natural History Apr 1971 S.C. Mitchell S. C. S. B. Korones H. W. W. Berendes Within a prospective study of 56,109 total births, 457 youngsters have been found to have congenital heart disease. The overall incidence is 8.14/1000 total births, 8.0/1000 for the Negro and 8.3/1000 for the white. A specific lesion has been identified for each patient and lesion frequencies given for each class of patient, stillbirth, neonatal death, infant death, childhood death, and survivors. The percentage of autopsies was 93% in the stillbirths, 89% in the neonatal deaths, and 76% for those dying after 28 days of age. Of those classified as having definite congenital heart disease, 93% have been examined by a pediatric cardiologist. The average follow-up time for the 272 survivors is 3 years. Thirty-five per cent of patients with ventricular septal defect surviving more than 6 months had their lesion close spontaneously; one-half of the survivors with tetralogy of Fallot were "atypical," and essentially equal numbers of blacks and whites had all types of coarctation of the aorta in line with the study population, which is 47% black and 53% white. Epidemiology of congenital heart disease in the United States Apr 1994 AM HEART J Richard F. Gillum Data from the National Center for Health Statistics were examined to aid in assessing the burden of congenital heart disease in the United States. In 1979 through 1988 combined, 46,450 deaths were attributed to CHD, 26,319 occurring in the first year of life. Another 14,283 deaths were attributed to other congenital anomalies of the circulatory system. In 1988 through 1990 combined, there were > 300,000 hospital discharges with any diagnosis of CHD, with 2.7 million days of care provided. For infants aged < 1 year, the rate was 13.7 per 1000 live births per year. Another 242,049 discharges had any diagnosis of other congenital circulatory anomalies. In 1983 through 1985, > 700,000 persons reported a history of CHD. In 1989, heart malformations were reported on the birth certificate of 4621 live births in 45 states and the District of Columbia. In 1988 through 1990 there were an estimated 37,445 repairs of atrial and ventricular septa. In addition, 45,635 cardiac catheterizations were performed in patients aged < 5. Average annual percentage declines in infant mortality rates for the period 1979 through 1988 were consistent with an effect of improvements in surgical technique and intensive care. The average annual infant mortality rate for hypoplastic left heart syndrome increased 1.3%, and rates for five causes amenable to treatment such as transposition of great vessels declined. Infant mortality rates in nonmetropolitan areas were higher than in metropolitan areas, suggesting that limited access to care might be associated with mortality.(ABSTRACT TRUNCATED AT 250 WORDS) Neutrophil-mediated acute lung injury after extracorporeal perfusion Jun 1994 J THORAC CARDIOV SUR D Johnson Dorothy J Thomson T Hurst Irvin Mayers A pulmonary injury of varying severity occurs routinely after cardiopulmonary bypass. We studied the pulmonary complications of partial cardiopulmonary bypass in four groups of dogs to better define the injury and to evaluate the efficacy of two interventions (addition of a leukocyte filter or cyclooxygenase inhibition) on preservation of systemic oxygenation. All animals received a standard anesthetic (pentobarbital, morphine, and vecuronium) and, after sternotomy, three groups of animals received 3 hours of partial cardiopulmonary bypass. The animals were randomized to receive partial bypass alone (n = 6), indomethacin and bypass (n = 5), or a leukocyte filter and bypass (n = 5). A fourth group (n = 5) did not receive bypass and served as a time control. We measured blood gases and also obtained histologic samples to assess the degree of lung injury. We found that bypass alone caused a significant reduction (p < 0.05) in arterial oxygen tension 1 hour after the conclusion of bypass (175 +/- 53 mm Hg) compared with prebypass values (357 +/- 41 mm Hg). Pretreatment with indomethacin ameliorated the decrease in arterial oxygen tension from prebypass to postbypass values (477 +/- 50 mm Hg versus 339 +/- 57 mm Hg, respectively). Similarly use of a leukocyte filter reduced the decline in arterial oxygen tension from prebypass to postbypass values (440 +/- 71 mm Hg versus 311 +/- 73 mm Hg, respectively). We believe that indomethacin ameliorates the decline in systemic oxygenation associated with bypass by augmentation of hypoxic pulmonary vasoconstriction and that the leukocyte filter acted to reduce pulmonary edema and thereby minimized intrapulmonary shunt. Atelectasis is a major cause of hypoxemia and shunt after cardiopulmonary bypass: an experimental study Nov 1997 ANESTHESIOLOGY Lennart Magnusson Vitas Zemgulis Stephan Wicky van Doyer Goran Hedenstierna Respiratory failure after cardiopulmonary bypass (CPB) remains a major complication after cardiac surgery. The authors tested the hypothesis that atelectasis is an important factor responsible for the increase in intrapulmonary shunt after CPB. Six pigs received standard CPB (bypass group). Six other pigs had the same surgery but without CPB (sternotomy group). Another six pigs were anesthetized for the same duration but without any surgery (control group). The ventilation-perfusion distribution was measured with the inert gases technique, extravascular lung water was quantified by the double-indicator distribution technique, and atelectasis was analyzed by computed tomography. Intrapulmonary shunt increased markedly after bypass but was unchanged over time in the control group (17.9 +/- 6.2% vs. 3.5 +/- 1.2%; P < 0.0001). Shunt also increased in the sternotomy group (10 +/- 2.6%; P < 0.01 compared with baseline) but was significantly lower than in the bypass group (P < 0.01). Extravascular lung water was not significantly altered in any group. The pigs in the bypass group showed extensive atelectasis (32.3 +/- 28.7%), which was significantly larger than in the two other groups. The pigs in the sternotomy group showed less atelectasis (4.1 +/- 1.9%) but still more (P < 0.05) than the controls (1.1 +/- 1.6%). There was good correlation between shunt and atelectasis when all data were pooled (R2 = 0.67; P < 0.0001). Atelectasis is produced to a much larger extent after CPB than after anesthesia alone or with sternotomy and it explains most of the marked post-CPB increase in shunt and hypoxemia. Surgery per se contributes to a lesser extent to postoperative atelectasis and gas exchange impairment. Challenges Posed by Adults With Repaired Congenital Heart Disease Jun 2001 CIRCULATION Joseph K. Perloff Carole A Warnes “Pediatric Congenital Cardiac Becomes a Postoperative Adult: The Changing Population of Congenital Heart Disease,” which was written in 1973,1 was the first publication devoted to what was destined to become a new field of special cardiovascular interest. Subsequent decades witnessed the maturity of this new subspecialty, which was formally recognized at the 22nd Bethesda Conference in 1990.2 It is altogether fitting for Circulation to include the present article in “Clinical Cardiology: New Frontiers.” The present article can be considered a sequel to the 1973 article, which dealt with the types of surgical interventions, the effects of those interventions on survival patterns, the desirability of primary anatomic repair in infancy, and the importance of postoperative residua and sequelae.1 The 1973 article opened by stating: “It is now possible to perform palliative or corrective surgery on almost all congenital cardiac anomalies, even the most complex … Survival patterns are affected, often profoundly. We are therefore confronted with a changing population of congenital cardiacs … However, we are obliged to look beyond the present and define our ultimate goal. What do we seek to accomplish? The answer is clear. Our efforts should focus on the quality of long-term survival.”1 Long-term survival is chiefly concerned with the growing number of postoperative patients who require continuing medical surveillance.3 What is needed is a new generation of cardiologists with a career interest in adult congenital heart disease; these highly trained specialists function best within tertiary care facilities designed for the comprehensive care of adult patients. With few exceptions, such facilities are part of major university hospital systems. Major facilities draw patients regionally, nationally, and internationally. The geographic distribution of tertiary care centers will best be determined by the quality of the facility and the services offered rather than by external constraints … Continuous perfusion of pulmonary arteries during total cardiopulmonary bypass favorably affects levels of circulating adhesion molecules and lung function Sep 2001 J THORAC CARDIOV SUR Takaaki Suzuki Tsutomu Ito Isamu Kashima Toyoki Fukuda Lung injury is a serious complication of cardiopulmonary bypass in infants with congenital heart disease and pulmonary hypertension. Cessation of blood flow in the pulmonary arteries during cardiopulmonary bypass is known to provoke lung dysfunction. We assessed the effect of continuous pulmonary perfusion on circulating adhesion molecules and on lung function. Fourteen infants with congenital heart disease and pulmonary hypertension were enrolled in the study. During total cardiopulmonary bypass, 8 patients underwent continuous perfusion of the pulmonary arteries (perfusion group), and the remaining 6 patients did not (control group). Plasma levels of circulating intercellular adhesion molecule 1, soluble granule membrane protein 140, and sialyl Lewis(x) and PaO (2)/fraction of inspired oxygen ratios were measured before commencement and serially for 24 hours after termination of bypass. Plasma levels of circulating intercellular adhesion molecule 1 decreased significantly at the termination of bypass in both groups but returned to prebypass levels immediately in the control group, whereas in the perfusion group the values remained significantly less than those before bypass. Plasma levels of soluble granule membrane protein 140 in the control group were significantly higher at 6 and 12 hours after bypass than levels before bypass, whereas in the perfusion group the values remained at the prebypass level throughout the postbypass period. Trends of plasma levels of sialyl Lewis(x) were alike in both groups. PaO (2)/fraction of inspired oxygen ratios in the control group decreased significantly from 6 hours after bypass, whereas values in the perfusion group remained at the prebypass value throughout the postbypass period. This study suggests that in infants having congenital heart disease and pulmonary hypertension, continuous pulmonary perfusion during total cardiopulmonary bypass minimizes ischemic insult and neutrophil-endothelial interaction mediated by adhesion molecules in the pulmonary microvessels. The Incidence of Congenital Heart Disease Jul 2002 J AM COLL CARDIOL Julien Hoffman Samuel Kaplan This study was designed to determine the reasons for the variability of the incidence of congenital heart disease (CHD), estimate its true value and provide data about the incidence of specific major forms of CHD. The incidence of CHD in different studies varies from about 4/1,000 to 50/1,000 live births. The relative frequency of different major forms of CHD also differs greatly from study to study. In addition, another 20/1,000 live births have bicuspid aortic valves, isolated anomalous lobar pulmonary veins or a silent patent ductus arteriosus. The incidences reported in 62 studies published after 1955 were examined. Attention was paid to the ways in which the studies were conducted, with special reference to the increased use of echocardiography in the neonatal nursery. The total incidence of CHD was related to the relative frequency of ventricular septal defects (VSDs), the most common type of CHD. The incidences of individual major forms of CHD were determined from 44 studies. The incidence of CHD depends primarily on the number of small VSDs included in the series, and this number in turn depends upon how early the diagnosis is made. If major forms of CHD are stratified into trivial, moderate and severe categories, the variation in incidence depends mainly on the number of trivial lesions included. The incidence of moderate and severe forms of CHD is about 6/1,000 live births (19/1,000 live births if the potentially serious bicuspid aortic valve is included), and of all forms increases to 75/1,000 live births if tiny muscular VSDs present at birth and other trivial lesions are included. Given the causes of variation, there is no evidence for differences in incidence in different countries or times. Clinical Respiratory Physiology of the Neonate and Infant With Congenital Heart Disease Feb 2004 Int Anesthesiol Clin Marcos Melo Mild Hypothermia Ameliorates Lung Ischemia Reperfusion Injury in an ex vivo Rat Lung Model Nov 2005 Tsuyoshi Shoji Mitsugu Omasa T. Nakamura Hiromi Wada Ischemia reperfusion (I-R) injury of the lung frequently occurs after cardiopulmonary bypass, pulmonary thromboendarterectomy, lung transplantation, and major pulmonary resection with vascular reconstruction. Mild hypothermia ameliorates ischemia reperfusion injury of the brain and the liver. However, the effect of mild hypothermia on I-R injury of the lung has not been investigated. The lungs of Lewis rats underwent 80 min of ischemia followed by 60 min of reperfusion in an ex vivo perfusion model. The ambient temperature was maintained at either normothermia (38 degrees C, n=6) or mild hypothermia (35 degrees C, n=6) during the ischemia and reperfusion. Pulmonary shunt fraction, peak inspiratory pressure, mean pulmonary arterial pressure during reperfusion, and the wet/dry weight ratio of the lung tissue at the end of reperfusion in the mild hypothermia group were significantly (p<0.05) lower than those in the normothermia group. Total adenine nucleotide, adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate after reperfusion in the mild hypothermia group were significantly (p<0.05) higher than those in the normothermia group. Mild hypothermia attenuates I-R injury of the lung with maintained levels of intrapulmonary high-energy phosphate compounds after reperfusion, suggesting its beneficial effect on warm lung I-R in clinical settings. Effects of ulinastatin treatment on the cardiopulmonary bypass-induced hemodynamic instability and pulmonary dysfunction May 2006 CRIT CARE MED Kazuhiro Nakanishi Shinhiro Takeda Atsuhiro Sakamoto Akira Kitamura To examine the association between decreased release of proinflammatory cytokines in response to urinary trypsin inhibitor pretreatment and decreased myocardial and lung injury after cardiopulmonary bypass. A prospective, randomized, double-blind study. University hospital. Thirty patients on cardiopulmonary bypass undergoing coronary artery bypass grafting. Patients received 5000 units/kg intravenous urinary trypsin inhibitor (n = 15) or 0.9% saline (control, n = 15) immediately before aortic cannulation for cardiopulmonary bypass. Neutrophil elastase, tumor necrosis factor-alpha, interleukin-6, and interleukin-8 were measured after intubation (T1), immediately before aortic cannulation (T2), after separation from cardiopulmonary bypass (T3), at the end of surgery (T4), and on postoperative days 1 (T5), 3 (T6), and 5 (T7). Simultaneous hematocrit values were obtained at all sample times. Isoenzyme of creatine kinase with muscle and brain subunits, troponin-T, and myosin light chain I were also measured. Various hemodynamic and pulmonary data were obtained perioperatively. Levels of neutrophil elastase and cytokines were corrected for hemodilution. Interleukin-6 and interleukin-8 levels were lower at T3 and T4 in the urinary trypsin inhibitor group than in the control group. Stroke volume index was significantly decreased in the control group at T3, and statistical difference was found between groups at T3 (p < .01). Respiratory index and intrapulmonary shunt were significantly higher in the control group than in the urinary trypsin inhibitor group at T3. Changes in respiratory index and intrapulmonary shunt correlated with interleukin-8 levels at T3 (r = .52, p < 00001; r = .37, p < 0001, respectively) and T4 (r = .44, p < .001; r = .24, p < .05, respectively). Neutrophil elastase levels and cardiac marker responses to coronary artery bypass grafting surgery were similar in both groups. Prepump administration of urinary trypsin inhibitor attenuates the elevation of interleukin-6 and interleukin-8 release immediately after cardiopulmonary bypass. Characterizing the Inflammatory Response to Cardiopulmonary Bypass in Children Jul 2006 Deborah Kozik James Tweddell Cardiopulmonary bypass is known to trigger a global inflammatory response. Age-dependent differences in the inflammatory response, the increased susceptibility to injury of immature organ systems, and the larger extracorporeal circuit to patient size ratio results in greater susceptibility of younger and smaller patients to the damaging effects of cardiopulmonary bypass. In this review the components of the inflammatory response to cardiopulmonary bypass are reviewed with special reference to the pediatric age group, including the age-specific impact on organ systems. In addition the current and evolving strategies to prevent, limit, and treat the inflammatory response to cardiopulmonary bypass in children are examined. View Sevoflurane for sedation in acute respiratory distress syndrome. A randomized controlled pilot study Jan 2017 792 Jabaudon Sevoflurane for sedation in acute respiratory distress syndrome. A randomized controlled pilot study. Jan 2017 792-800 Jabaudon M Boucher P Imhoff E Sehua Qu Lianqiang Shan Xin Chen Huaifu Dong Background This study aimed to determine whether postnatal treatment with recombinant human IGF-1 (rhIGF-1)/binding peptide 3 (BP3) ameliorates lung injury and prevents pulmonary hypertension (PH) in bronchopulmonary dysplasia (BPD) models. Methods We used two models of BPD in this study: one model that was associated with chorioamnionitis (CA), stimulated by intra-amniotic fluid and exposure to ... [Show full abstract] lipopolysaccharide (LPS), whereas the other was exposed to postnatal hyperoxia. Newborn rats were treated with rhIGF-1/BP3 (0.2 mg/Kg/d) or saline via intraperitoneal injection. The study endpoints included the wet/dry weight (W/D) ratio of lung tissues, radial alveolar counts (RACs), vessel density, right ventricular hypertrophy (RVH), lung resistance, and lung compliance. Hematoxylin and eosin (H&E) and Masson staining were used to evaluate the degree of lung injury and pulmonary fibrosis. IGF-1 and eNOS expression were detected using western blotting or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The levels of SP-C, E-cadherin, N-cadherin, FSP1, and Vimentin in the lung tissues were detected by immunofluorescence. Results LPS and hyperoxia treatment increased lung injury and pulmonary fibrosis, enhanced RVH and total respiratory resistance, and decreased RAC, pulmonary vascular density and pulmonary compliance in young mice (all p < 0.01). Simultaneously, LPS and hyperoxia induced an increase in epithelial-mesenchymal transition (EMT) in airway epithelial cells. However, rhIGF-1/BP3 treatment reduced lung injury and pulmonary fibrosis, decreased RVH and total respiratory resistance, and enhanced RAC, pulmonary vascular density and pulmonary compliance, as well as inhibited EMT in airway epithelial cells in LPS and hyperoxia treated mice. Conclusion Postnatal rhIGF-1/BP3 treatment relieved the effects of LPS or hyperoxia on lung injury and prevented RVH, providing a promising strategy for the treatment of BPD.	https://www.researchgate.net/publication/351743749_The_Characterization_of_Postoperative_Mechanical_Respiratory_Requirement_in_Neonates_and_Infants_Undergoing_Cardiac_Surgery_on_Cardiopulmonary_Bypass_in_a_Single_Tertiary_Institution
Trace-level determination and insight in speciation of silicon in petrochemical samples by flow-injection high resolution ICP MS and HPLC-high resolution ICP MS \| Request PDF Request PDF \| Trace-level determination and insight in speciation of silicon in petrochemical samples by flow-injection high resolution ICP MS and HPLC-high resolution ICP MS \| A method for the determination of silicon in organic solutions was developed using a double focusing magnetic sectorICP MS operating at a... \| Find, read and cite all the research you need on ResearchGate Trace-level determination and insight in speciation of silicon in petrochemical samples by flow-injection high resolution ICP MS and HPLC-high resolution ICP MS September 2010 Journal of Analytical Atomic Spectrometry 25(9) DOI: 10.1039/c005010e Abstract A method for the determination of silicon in organic solutions was developed using a double focusing magnetic sectorICP MS operating at a resolution of 4000. The signal-to-background ratio was improved 10-fold over standard sample introduction systems by the use of micro-flow injection total consumption sample introduction. The detection limits down to 1 ng g−1, the calibration graph linear over 4 orders of magnitude and precision to within a few percent were obtained. The effects of the sample matrix and of the chemical form of silicon on the sensitivity were investigated and alleviated when necessary by heating the spray chamber and sample dilution. Normal phase HPLC-ICP MS and size-exclusion ICP MS were proposed to gain an insight into the purity of the silicon standard compounds, their reactivity with different petroleum-related matrices and speciation of silicon. ... It is often added to oils and fuels to prevent foam and emulsion formation. Silicon reacts with hydro-desulfurization and hydro-demetallization catalysts and poisons them [3] . The deposition of silicon in the engine sensors may cause wrong sensing in the fuel management system. ... ... Up to now, different methods including anodic stripping voltammetry (ASV) [2], FIA-ICP-MS [3] , electrothermal atomic absorption spectrometry (ET-AAS) [4], DRC-ICP-MS [5], UV-Vis [6], X-ray fluorescence (XRF) [7], continuum source flame atomic absorption spectrometry [8], capillary electrophoresis [9], and reversed-phase ion-pair chromatography [10] have been developed for silicon determination. Each technique has advantages and suffers from some shortcomings. ... ... A mercury solution (10 mM) were prepared from Hg(NO 3 ) 2 in deionized water. A stock solution of silicon was prepared from the atomic absorption standard solution of silicon (1000 ppm). ... Bifunctional Nitrogen and Fluorine Co‐doped Carbon Dots as Fluorescence Probe for Silicon and Mercury by pH Switching New nitrogen and fluorine co-doped carbon dots were synthesized and used as a dual function fluorescent probe for silicon and mercury ions. The size of CDs was 10 nm. At optimum conditions (pH = 13, λex = 360 nm, and λem = 518 nm), the detection limit (DL) of silicon was 16.6 nM. Linear calibration was observed in the range of 0.8–35 µM. This fluorescence probe for silicon detection is presented for the first time and had the lowest detection limit in comparison with different previously reported techniques. In addition to the above property, these co-doped carbon dots had the second function as a fluorescence probe for mercury detection at pH = 8. The DL for mercury was 38 nM. The performance of this probe was also compared with other co-doped carbon dots. Excellent sensitivity and selectivity, simple method, low-cost materials, and applicability in real sample analysis are advantages of this dual function fluorescence probe. ... Since their commercial introduction in 1943, the production of siloxanes and polydimethylsiloxanes has been highly developed, and currently, there is wide application of the numerous materials and products of this industry including coatings, textiles, rubbers, seals, insulating materials, ceramics, greases, waxes, implants, additives, cosmetics and consumer goods [1][2]. Silicon is also present in crude oil and petroleum products (0.001-100 mg kg −1 ) [1, [10] [11][12][13][14][15][16][17][18], plants (0.01-20% of dry mass) [2,4,[18][19][20], water (1-600 mg L − 1 ) [2,4], animal bodies (0.01-40%) [1][2]4,21], human tissues (4-200 mg kg −1 ) [1][2]4,22], and materials from the electronics or polymer industries [1][2]23,24]. ... ... Various analytical techniques have been employed for Si determination, including gravimetry, volumetry, UV-VIS spectrometry, neutron activation analysis and X-ray spectrometry [1,2]. For silicon determination at trace levels, the most commonly used atomic spectrometry techniques include inductively coupled plasma mass spectrometry (ICPMS) [10] [11][12][13][14]22], inductively coupled plasma optical emission spectrometry (ICP-OES) [14,25], microwave-induced plasma optical emission spectrometry [18], graphite furnace atomic absorption spectrometry (GFAAS) [8,17,21,23,24] and flame atomic absorption spectrometry (FAAS) [15][16]19]. ... ... Several other important drawbacks are the special difficulties arising from particular analytical techniques. In the case of ICP MS, severe spectral interferences coming from polyatomic ions disturb the determination of all Si isotopes [1,2,7, [10] [11][12][13]22]. Another difficulty is the dependence of the signal on the chemical form of the analyte when the sample is delivered to inductively coupled plasma [12,14,25]. ... Spectral aspects of the determination of Si in organic and aqueous solutions using high-resolution continuum source or line source flame atomic absorption spectrometry Apr 2016 SPECTROCHIM ACTA B Zofia Kowalewska Janusz Pilarczyk Łukasz Gościniak High-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS) was applied to reveal and investigate spectral interference in the determination of Si. An intensive structured background was observed in the analysis of both aqueous and xylene solutions containing S compounds. This background was attributed to absorption by the CS molecule formed in the N2O–C2H2 flame. The lines of the CS spectrum at least partially overlap all five of the most sensitive Si lines investigated. The 251.611 nm Si line was demonstrated to be the most advantageous. The intensity of the structured background caused by the CS molecule significantly depends on the chemical form of S in the solution and is the highest for the most-volatile CS2. The presence of O atoms in an initial S molecule can diminish the formation of CS. To overcome this S effect, various modes of baseline fitting and background correction were evaluated, including iterative background correction (IBC) and utilization of correction pixels (WRC). These modes were used either independently or in conjunction with least squares background correction (LSBC). The IBC + LSBC mode can correct the extremely strong interference caused by CS2 at an S concentration of 5% w:w in the investigated solution. However, the efficiency of this mode depends on the similarity of the processed spectra and the correction spectra in terms of intensity and in additional effects, such as a sloping baseline. In the vicinity of the Si line, three lines of V were recorded. These lines are well-separated in the HR-CS FAAS spectrum, but they could be a potential source of overcorrection when using line source flame atomic absorption spectrometry (LS FAAS). The expected signal for the 251.625 nm Fe line was not registered at 200 mg L− 1 Fe concentration in the solution, probably due to the diminished population of Fe atoms in the high-temperature flame used. The observations made using HR-CS FAAS helped to establish a “safe” level of the matrix components S, V, and Fe in the determination of Si using both HR-CS FAAS and LS FAAS. Under the best conditions, the instrumental detection limits for LS FAAS and for HR-CS FAAS, as well as the characteristic concentration values, were similar. Unusual calibration curves were observed in HR-CS FAAS. The curves showed an inflexion point that divided them into two linear parts with distinctly different slopes. ... Due to the initial injection of PDMS generally about 50 mg/kg, silicon compounds are produced at trace levels in petroleum products. Total silicon content can be measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) [7] and ICP-MS [8] . The concentrations generally reported range from several hundred μg of Si/kg to several mg of Si/kg in petroleum products [3]. ... ... Three distributions of silicon compounds with masses ranging from 150 to 116,500 g/mol were quantified with a limit of quantification (LOQ) of 1 mg of Si/kg in PDMS degradation samples [15]. More recently, HPLC (high pressure liquid chromatography) combined to ICP-HRMS was applied for the speciation of standard silicon compounds in xylene [8] . Pure products commercially available were not totally eluted with NP (normal phase)-HPLC and the method cannot be applied for silicon speciation in petroleum products with these chromatographic conditions without any further developments. ... ... The ICP-MS response is linear with correlation coefficients up to 0.99 but it is not universal for all silicon compounds. The non-universal response was previously demonstrated during total silicon analysis by ICP-OES [7, 8, [28][29][30]. This problem was solved by Sanchez et al. [31] using a TISIS (total integrated samples introduction system) heated at 350°C to minimize matrix interferences observed in various petroleum products and to suppress difference on silicon response by ICP-OES. ... Towards silicon speciation in light petroleum products using gas chromatography coupled to inductively coupled plasma mass spectrometry equipped with a dynamic reaction cell SPECTROCHIM ACTA B Fabien Chainet Charles-Philippe Lienemann Jérémie Ponthus Olivier F. X. Donard Silicon speciation has recently gained interest in the oil and gas industry due to the significant poisoning problems caused by silicon on hydrotreatment catalysts. The poisoning effect clearly depends on the structure of the silicon species which must be determined and quantified. The hyphenation of gas chromatography (GC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) allows a specific detection to determine the retention times of all silicon species. The aim of this work is to determine the retention indices of unknown silicon species to allow their characterization by a multi technical approach in order to access to their chemical structure. The optimization of the dynamic reaction cell (DRC) of the ICP-MS using hydrogen as reactant gas successfully demonstrated the resolution of the interferences (14N14N+ and 12C16O+) initially present on 28Si. The linearity was excellent for silicon compounds and instrumental detection limits ranged from 20 to 140 μg of Si/kg depending on the response of the silicon compounds. A continuous release of silicon in the torch was observed most likely due to the use of a torch and an injector which was made of quartz. A non universal response for silicon was observed and it was clearly necessary to use response coefficients to quantify silicon compounds. Known silicon compounds such as cyclic siloxanes (D3-D16) coming from PDMS degradation were confirmed. Furthermore, more than 10 new silicon species never characterized before in petroleum products were highlighted in polydimethylsiloxanes (PDMS) degradation samples produced under thermal cracking of hydrocarbons. These silicon species mainly consisted of linear and cyclic structures containing reactive functions such as ethoxy, peroxide and hydroxy groups which can be able to react with the alumina surface and hence, poison the catalyst. This characterization will further allow the development of innovative solutions such as trapping silicon compounds or strengthening the resistivity of the hydrotreatment catalyst against silicon. ... The presence of silicon in petroleum products originates in the use of polydimethylsiloxanes (PDMS) which are introduced to enhance the crude oil recovery from the reservoir and as antifoaming agents to avoid emulsion phenomena in the different processes such as coking, visbreaking, steam cracking or distillation [6,7]. The total silicon concentration determined by ICP-OES [8] or by ICP/MS [9] was ranging from several hundred lg/kg to some several mg/kg. PDMS degrades around 300°C [10][11][12][13] despite its rather good thermal stability. ... ... (Methylpropyl) cyclic siloxanes (12) have been also synthesized and characterized using GC by Youdina et al. [44] and Hayden and Barlow [45]. Similarly to the ethoxy methyl cyclic siloxanes (5) and di(ethoxy methyl) cyclic siloxane (9) , the chemical structure of the silicon species family 12 could be potentially explained by the reactivity of carbon radicals with PDMS degradation reaction products. ... Characterization of silicon species issued from PDMS degradation under thermal cracking of hydrocarbons: Part 2 -Liquid samples analysis by a multi-technical approach based on gas chromatography and mass spectrometry Article Jan 2014 FUEL Fabien Chainet Le Meur L. Charles-Philippe Lienemann Donard O.F.X Silicon speciation has recently gained interest in the oil and gas industry due to the impact of silicon species on hydrotreatment (HDT) catalysts. The determination of the chemical structure of silicon compounds appears as essential to limit silicon poisoning as to improve the lifetime of catalysts. To achieve a representative speciation of silicon in petroleum products, fresh samples of PDMS degradation under thermal cracking of hydrocarbons were produced using a pilot plant. The samples were carefully stored in a dewar containing liquid nitrogen (−195 °C) to minimize the possible evolution of silicon species and to allow their analysis in representative conditions. A complete analytical approach based on gas chromatography (GC) and mass spectrometry techniques (MS) was developed and applied to the samples produced at 500 °C. Moreover, to resolve coelutions observed by GC/TOFMS, GC-GC/TOFMS was successfully applied to obtain the mass spectrum of only one silicon compound. Combining the GC/MS mass spectrum giving access to the fragmentation of the compound and the raw formula and double bond equivalent (DBE) obtained by ESI-FT-ICR/MS, chemical structures were proposed. Almost molecules were strengthened by MSn. View ... Speciation analysis is a sophisticated analytical method, which can provide qualitative and quantitative information about the species that allow the identification and classification of metalloporphyrins in crude oil. Several methods have been proposed for this purpose, such as laser desorption ionization time-of-flight mass spectrometry (LDI-TOF-MS) [14], size exclusion chromatography hyphenated to inductively coupled plasma mass spectrometry (SEC-ICP-MS) [9,15], high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) [12, 16, 17], liquid chromatography hyphenated to high resolution mass spectrometry (LC-HRMS) [18], gas chromatography hyphenated to mass spectrometry (GC-MS) [19,20], Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) [21], X-ray fluorescence (XRF) [22] and non-chromatographic methods based on graphite furnace atomic absorption spectrometry (GF AAS) [3,[23][24][25]. ... Non-chromatographic method for separation and determination of Fe, Ni and V porphyrins in crude oil Feb 2019 TALANTA Maciel Luz Pedro Oliveira A method combining ultracentrifugation and cloud point extraction is proposed for the separation and determination of Fe, Ni and V porphyrins in crude oil. An emulsion containing about 200 mg of crude oil, 6% (w v ⁻¹ ) of Triton X-100™, 1.6% (v v ⁻¹ ) of chloroform and 0.1 mol L ⁻¹ of HCl was prepared. An aliquot (1.5 mL) of this emulsion was ultracentrifuged to separate the crude oil heavy fraction (asphaltene and particulate matter) and inorganic species, organometallic compounds and porphyrins of Fe, Ni and V remained in the supernatant. Another aliquot (1.5 mL) was heated up to 90 °C for 5 min to separate the porphyrins, both heavy and light organics compounds, light organic and particulate matter by cloud point extraction, only the inorganic and ionic organometallic compounds species remained in the aqueous phase. The supernatant and aqueous phase were analysed by simultaneous graphite furnace atomic absorption spectrometry. The concentration determined in the supernatant was subtracted from that determined in the aqueous phase, providing the quantification of Fe, Ni and V porphyrin in the crude sample. The accuracy of the proposed method was checked using SARA reference method. The elemental determinations in the fractions obtained using the proposed method and the comparison by SARA showed an absence of Fe in the aromatics and resins fractions. The results showed that about 40% of Ni and 70% of V in the crude oil sample are bounded to porphyrins. ... The presence of silicon in petroleum fractions is caused by polydimethylsiloxanes (PDMS), which are added to increase the recovery of crude oil from the reservoir and as defoamers to avoid foaming in various processes, such as coking, visbreaking, steam cracking or distillation [1,2]. Total concentration of silicon can vary from a few hundred μg/kg to several mg/kg [3, 4] . As it was shown in [5], thermal decomposition of PDMS results in the formation of cyclic siloxanes as the major products. ... Guard Bed Catalysts for Silicon Removal During Hydrotreating of Middle Distillates Article Dec 2018 CATAL TODAY K. A. Nadeina Maxim Kazakov A.A. Kovalskaya A. S. Noskov The influence of textural characteristics of NiMo/Al2O3 on silicon capacity and hydrotreating activity has been investigated so as to develop guard-bed catalyst for silicon removal from middle distillates. Series of NiMo/Al2O3 catalysts with a specific surface area from 150 to 210 m²/g and an average pore diameter from 21.1 to 10.5 nm have been synthesized. The difference in textural characteristics has been achieved by variation of aging time and temperature of hydrothermal treatment of boehmites. The boehmites, supports and catalysts were studied by various physico-chemical methods: XRD, nitrogen adsorption-desorption, IR spectroscopy, UV–vis, ¹H NMR, ²⁷Al NMR, XPS, SEM, HRTEM. Catalysts were tested in hydrotreating of diesel fuel contaminated with decamethylcyclopentasiloxane as a model silicon compound. It is shown that the increase in aging time and temperature of hydrothermal treatment of initial boehmites results in the decrease of OH groups concentration per 1 g of an alumina support. The increase of the OH groups content leads to higher silicon capacity, while HDS activity of catalysts decreases. The study of catalysts surface after reaction showed that Si located on a support surface and did not cover directly active component particles. However, its location on the support near edges of active component particles could result in the decrease of HDS activity due to limited accessibility of the active sites. ... Regarding the importance of Si for many biological life systems, several analytical techniques for Si determination were reported. Among them, graphite furnace atomic absorption spectrometry (GF AAS) [1,13,14], inductively coupled plasma optical emission spectrometry (ICP OES) [15,16], inductively coupled plasma mass spectrometry (ICP-MS) [17] , laser-induced breakdown spectroscopy (LIBS) [18] and X-ray fluorescence spectrometry (XRF) [19,20] were employed for biological, inorganic and plant materials analysis. Nevertheless, even though excellent limits of detection were achieved, some of these techniques are costly and require sample pretreatment to provide total matrix decomposition. ... Determination of silicon in plant materials using direct solid sample analysis with high-resolution continuum source graphite furnace atomic absorption spectrometry Article Oct 2015 MICROCHEM J Wiliam Boschetti Luiza Dalagnol Mariana Dullius Jailson B. de Andrade ... Speciation analysis is a sophisticated analytical method, which can provide qualitative and quantitative information about the species that allow the identification and classification of metalloporphyrins in crude oil. Several methods have been proposed for this purpose, such as laser desorption ionization time-of-flight mass spectrometry (LDI-TOF-MS) [14], size exclusion chromatography hyphenated to inductively coupled plasma mass spectrometry (SEC-ICP-MS) [9,15], high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) [12, 16, 17], liquid chromatography hyphenated to high resolution mass spectrometry (LC-HRMS) [18], gas chromatography hyphenated to mass spectrometry (GC-MS) [19,20], Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) [21], X-ray fluorescence (XRF) [22] and non-chromatographic methods based on graphite furnace atomic absorption spectrometry (GF AAS) [3,[23][24][25]. ... Fast emulsion-based method for simultaneous determination of Co, Cu, Pb and Se in crude oil, gasoline and diesel by graphite furnace atomic absorption spectrometry Full-text available Oct 2013 Maciel Luz Angerson Nogueira do Nascimento Pedro Oliveira ... High resolution ICP-MS, isotopic dilution and collision/reaction cells have been the most efficient strategies to overcome these difficulties [15]. Pohl et al., for example, determined Si in organic solutions and petrochemical samples using a double focusing magnetic sector high resolution ICP-MS [16] . ... A simple dilute-and-shoot procedure for Si determination in diesel and biodiesel by microwave-induced plasma optical emission spectrometry Article Jan 2013 MICROCHEM J Renata S. Amais George L. Donati Daniela Schiavo Joaquim A. Nóbrega In the present work, microwave-induced plasma optical emission spectrometry (MIP OES) and the flow blurring nebulizer (FBN) technology are used to determine silicon in diesel and biodiesel samples. A simple dilute-and-shoot procedure with ethanol is presented. Two additional sample preparation procedures are also evaluated for comparison: closed-vessel microwave-assisted acid digestion and microemulsion preparation in n-propanol. Limits of detection (LOD) vary from 5 to 20 μg L− 1 and relative standard deviations (RSD) are lower than 2% in all cases. Accuracy is evaluated by spike experiments, with recoveries between 80 and 103%. The influence of Ca, K, Mg and Na as concomitant ions on silicon determination by MIP OES is also investigated. Significant effects on Si emission signals are observed neither in single nor multiple concomitant experiments. Although good results are obtained with all sample preparation procedures evaluated, sample dilution in ethanol represents a simpler and faster strategy, which allows external calibration with inorganic standards and aqueous solutions. Past to Future: Application of Gel Permeation Chromatography from Petroleomics and Metallopetroleomics to New Energies Applications: A Minireview Jun 2023 Wladimir Ruiz Rémi Moulian Ryan P. Rodgers Julie Guillemant Direct Determination of Dissolved Silicon in Atmospheric Precipitation by CH4−Ar Mixed Plasma DRC-ICP-MS Nov 2022 Zhang Jiangyi Zhifang Xu Is it possible to reactivate hydrotreating catalyst poisoned by silicon? Mar 2021 CATAL TODAY Yu. V. Vatutina Maxim Kazakov K. A. Nadeina A. S. Noskov The feasibility of the reactivation of CoMo/Al2O3 hydrotreating catalysts poisoned by Si compounds has been studied. CoMo/Al2O3 catalysts poisoned with 3, 4 and 5 wt.% of silicon were obtained during hydrotreating of diesel fraction contaminated with decamethylcyclopentasiloxane. Catalysts poisoned by different amount of silicon were regenerated by oxidative treatment and subsequently reactivated using citric acid solution. The catalysts were studied by nitrogen adsorption-desorption method, CHNS analysis, UV–Vis, thermal analysis, SEM, HRTEM, XPS. It was shown that the hydrodesulfurization activity of regenerated catalysts decreased with increasing silicon content. According to UV-Vis results, the increase in Si content on the spent catalyst leads to the formation of CoOx oxides after oxidative regeneration. Probably, cobalt oxides do not promote MoS2 slabs during sulfidation, convert to inactive Co species and decrease hydrodesulfurization activity. After reactivation procedure, there was the increase in active component particles dispersion, while catalytic activity in hydrodesulfurization of dibenzothiophene and hydrodenitrogenation of quinoline increased. It was established that hydrodesulfurization and hydrodenitrogenation activities of CoMo/Al2O3 catalyst with less than 3 wt.% of Si could be completely restored by reactivation. Silicon speciation in light petroleum products using gas chromatography coupled to ICP-MS/MS Article Aug 2020 Raquel Sánchez Fabien Chainet Vincent Souchon José Luís Todolí Silicon speciation in light petroleum products through the hyphenation of gas chromatography coupled to ICP–tandem mass spectrometry (GC-ICP-MS/MS) is described in the present work. Eleven silicon compounds (nine siloxanes, trimethylsilanol and triethylsilane) have been taken as model molecules. The carrier and optional gas flow rates as well as the hydrogen gas flow rate in the octopole reaction cell (ORC) were critical variables. They precluded both the sensitivity and the extent of the effects caused by the sample matrix and silicon nature. The optimization of the latter variable mitigated the m/z 28 interference (¹⁴N¹⁴N⁺ and 12C16O⁺). Moreover, under the optimized conditions, a universal response was found irrespectively of the silicon chemical form and sample matrix. The analytical performances of the method have been evaluated. Thus, the ICP-MS/MS response was linear between 0 to 500 µg kg⁻¹ with correlation coefficients up to 0.999, whereas the method quantification limit (MQL) ranged from 8 to 60 μg kg⁻¹. Moreover, no drift was found for quality control samples analysed along a given analytical run. Only, the most volatile compound, i.e., trimethylsilanol (TMSOH), induced a drop in sensitivity caused by its partial evaporation from the GC vial. The method was validated by the analysis of real samples and the results were compared with those obtained by inductively coupled plasma optical emission spectrometry (ICP-OES) and X-rays fluorescence. No significant differences in Si total content provided by the three methods were found. Real light petroleum products, especially coker naphtha samples, were analyzed and it was verified that they only contained cyclic siloxanes (D3-D6), mainly hexamethylcyclotrisiloxane (D3) and octamethylcyclotetrasiloxane (D4). ACQUIRING INFORMATION ON TOTAL CONTENT AND SPECIATION OF SILICON IN PETROLEUM PRODUCTS/FUELS USING HIGH-RESOLUTION CONTINUUM SOURCE FLAME ATOMIC ABSORPTION SPECTROMETRY AND WAVELENGTH DISPERSIVE X-RAY FLUORESCENCE Dec 2018 Zofia Kowalewska Karolina Brzezińska Katarzyna Cikorska Łukasz Gościniak Various chemical forms of silicon (Si), possible present in petroleum products/fuels, were investigated in this work: low molecular mass species of different composition, structure and volatility as well as oil Conostan standard, polysiloxanes and silicon dioxide. Analysis in xylene solutions was accomplished using wavelength dispersive X-ray fluorescence (WD XRF) or high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS). Samples were also analysed, using HR-CS FAAS, in aqueous solutions after: mineralization in closed microwave heated system, mineralization in open system or the mineralization combined to fusion with lithium metaborate. It was found that none of the applied procedures was suitable for accurate determination of Si in all the tested forms. The total content of Si can be only known, if various methodological variants are applied. Using WD XRF similar signals are obtained for all the investigated organic Si forms. The procedure is also helpful to detect sedimentation (presence) of inorganic Si compounds (successive measurements in the same cup). In direct measurements of organic solutions using HR-CS FAAS analytical response is similar for all the investigated organic forms, apart from the significantly increased signal for the most volatile forms. On the other hand, underestimated results were stated due to losses of volatile and medium volatile Si compounds and/or difficulty in analyte transferring to an aqueous solution in the procedures comprising mineralization. The procedure combining mineralization and fusion gives quantitative results for SiO2, Conostan standard and some polysiloxanes. Similar behavior of Si compounds belonging to a given group (organic very volatile, medium volatile, non-volatile or inorganic) and leading to a systematic error in a given procedure was used to acquire knowledge on the presence of this kind of Si species in a sample. The proposed proceedings scheme, employs easy available analytical techniques and can be widely applied. It was found, that internal standard calibration, available in HR-CS FAAS, does not correct chemical interference due to the LiBO2 matrix. However, using vanadium (V) as internal standard, it is possible to correct sensitivity drift in time. Feasibility of high-resolution continuum source flame molecular absorption spectrometry for silicon determination in organic solutions via SiO molecule Jan 2018 Zofia Kowalewska In this work, for the first time, Si determination via absorption by its diatomic molecule has been proposed. The high-resolution continuum source flame molecular absorption spectrometry (HR-CS FMAS) has been... View Understanding the impact of silicon compounds on metallic catalysts through experiments and multi-technical analysis Jan 2017 CR CHIM Anne-Claire Dubreuil Fabien Chainet Rui Miguel de Sousa Bartolomeu Charles-Philippe Lienemann The presence of silicon in petroleum products is a major issue due to its poisoning effect on catalysts. The aim of this work is to combine silicon speciation and poisoning tests. Cyclic siloxanes were the main silicon species found in petroleum products. Other silicon compounds, comprising reactive groups (hydroxy, methoxy and hydroperoxy), were also recovered but at trace levels using GC-ICP/MS. Five well-chosen silicon compounds were used to poison Pd/alumina catalysts. Only dimethoxydimethylsilane poisons Pd-catalysts while polydimethylsiloxane (PDMS) has no effect on their activities in buta-1,3-diene hydrogenation. Unexpectedly, triethylsilane, triethylsilanol and even octamethylcyclotetrasiloxane (D4) exhibit a promoting effect. An interpretation of the phenomena based on various characterizations is proposed. Niobium carbide as permanent modifier for silicon determination in petrochemical products by emulsion-based sampling GF AAS Jan 2014 FUEL Maciel Luz Pedro Oliveira The sample preparation was carried out by emulsion-based sampling, using about 200 mg of samples (crude oil, diesel or gasoline), 125 μL of hexane (only for crude oil) and 7.5 mL of Triton X-100 (20% v v−1). The mixture was stirred manually for few seconds, submitted to ultrasound bath for 30 min, and diluted to 25 mL with high-purity water. The combination of NbC as permanent modifier to avoid the formation of thermally stable SiC and 20 μg of Pd as co-injected chemical modifier to avoid losses of volatile silicon was mandatory for the success of the proposed method. In these conditions, the sensitivity of silicon was increased around six times and the relative standard deviation was decreased around 10 times. After heating program optimization, the pyrolysis and atomization temperatures were 1300 °C and 2600 °C, respectively. The limit of detection, limit of quantification and characteristic mass were, respectively, 2.6 μg L−1, 8.6 μg L−1 and 141 pg. The relative standard deviation was lower than 1% for 100 μg L−1 of Si. The accuracy was checked by the analysis of oil-based Si standards and the found values are in accordance with the reference values at 95% confidence level. Addition of 30 μg L−1 of Si in crude oil, gasoline and diesel samples resulted in recoveries ranging from 95% to 111%. Applications of Nanoparticles in Elemental Speciation Feb 2015 Chuan-Ting Liu An-Na Tang Nanoparticles, notable for their small size, high surface area to volume ratios, and strong adsorption capacity, have been the subject of great interest in analytical chemistry. Over the past decade, nanoparticles have been widely used as adsorbents for elemental speciation. Elemental ions or chelates may interact with nanoparticles by van der Waals forces, hydrogen bonds, and hydrophobic interactions. Various types of nanoparticles used for elemental speciation, including magnetic nanoparticles, carbon nanotubes, metallic oxide nanoparticles, nanostructured mixed oxides, and ion imprinted polymer nanoparticles, are reviewed. Future trends and development in this research area are discussed. Environmental Chemistry of Organosiloxanes Dec 2014 Christoph Rücker Klaus Kümmerer Available production figures are rather diverse with respect to compound classes covered, inclusion or not so of intermediates, and geographical and temporal coverage. They are therefore difficult to compare, and some seem to be conflicting. The numbers given in the following nevertheless illustrate the order of magnitude and the dynamic increase of production and use of organosiloxanes. Taking into account the above uncertainties, these figures seem roughly consistent. Thus, the current annual worldwide production of organosiloxanes may reasonably be assumed to be several million tons. All of this material, after a shorter or longer time, will be released to the environment in one form or another. For example, silicones used in construction are conserved in a building for many years, to be released upon demolition, while other applications are based on shorter time scales. Determination of trace elements in petroleum products by inductively coupled plasma techniques: A critical review Full-text available Jun 2013 SPECTROCHIM ACTA B Raquel Sánchez José Luís Todolí Charles-Philippe Lienemann A solid-phase extraction (SPE) coupled with dispersive liquid–liquid microextraction based on the solidification of floating organic drop (DLLME-SFO) method has been developed for extraction and determination of organic bisphenol A (BPA) in different real samples. Bisphenol A was determined by high-performance liquid chromatography-fluorescence detector (HPLC-FL). Variables affecting the ... [Show full abstract] performance of both steps were thoroughly investigated. The analytical characteristics of the method were determined. The calibration graph is linear in the range of 0.005–10 ng g−1 with detection limit of 0.002 ng g−1. The relative standard deviation (RSD) for of BPA in different samples are in the range of 6.8–9.6 % (n = 5). The obtained results show that SPE-DLLME-SFO combined with HPLC-FL is a fast and simple method for the determination of BPA in food and environmental samples.	https://www.researchgate.net/publication/244554737_Trace-level_determination_and_insight_in_speciation_of_silicon_in_petrochemical_samples_by_flow-injection_high_resolution_ICP_MS_and_HPLC-high_resolution_ICP_MS
The Lancet Global Health The Lancet Global Health \| Citations: 457 \| Read 1100 articles with impact on ResearchGate, the professional network for scientists. The Lancet Global Health Print ISSN: 2214-109X Publications Annual rates of decline in child, maternal, HIV, and tuberculosis mortality across 109 countries of low and middle income from 1990 to 2013: An assessment of the feasibility of post-2015 goals Article Stéphane Verguet Ole F Norheim Zachary D Olson Dean T Jamison Background: Measuring a country's health performance has focused mostly on estimating levels of mortality. An alternative is to measure rates of decline in mortality, which are more sensitive to changes in health policy than are mortality levels. Historical rates of decline in mortality can also help test the feasibility of future health goals (eg, post-2015). We aimed to assess the annual rates of decline in under-5, maternal, tuberculosis, and HIV mortality over the past two decades for 109 low-income and middle-income countries. Methods: For the period 1990-2013, we estimated annual rates of decline in under-5 mortality (deaths per 1000 livebirths), the maternal mortality ratio (deaths per 100 000 livebirths), and tuberculosis and HIV mortality (deaths per 100 000 population per year) using published data from UNICEF and WHO. For every 5-year interval (eg, 1990-95), we defined performance as the size of the annual rate of decline for every mortality indicator. Subsequently, we tested the feasibility of post-2015 goals by estimating the year by which countries would achieve 2030 targets proposed by The Lancet's Commission on Investing in Health (ie, 20 deaths per 1000 for under-5 mortality, 94 deaths per 100 000 for maternal mortality, four deaths per 100 000 for tuberculosis mortality, and eight deaths per 100 000 for HIV mortality) at observed country and aspirational best-performer (90th percentile) rates. Findings: From 2005 to 2013, the mean annual rate of decline in under-5 mortality was 4·3% (95% uncertainty interval [UI] 3·9-4·6), for maternal mortality it was 3·3% (2·5-4·1), for tuberculosis mortality 4·1% (2·8-5·4), and for HIV mortality 2·2% (0·1-4·3); aspirational best-performer rates per year were 7·1% (6·8-7·5), 6·3% (5·5-7·1), 12·8% (11·5-14·1), and 15·3% (13·2-17·4), respectively. The top two country performers were Macedonia and South Africa for under-5 mortality, Belarus and Bulgaria for maternal mortality, Uzbekistan and Macedonia for tuberculosis mortality, and Namibia and Rwanda for HIV mortality. At aspirational rates of decline, The Lancet's Commission on Investing in Health target for under-5 mortality would be achieved by 50-64% of countries, 35-41% of countries would achieve the 2030 target for maternal mortality, 74-90% of countries would meet the goal for tuberculosis mortality, and 66-82% of countries would achieve the target for HIV mortality. Share National and regional estimates of term and preterm babies born small for gestational age in 138 low-income and middle-income countries in 2010 Article Full-text available Anne Cc Lee Joanne Katz Hannah Blencowe [...] Robert E Black National estimates for the numbers of babies born small for gestational age and the comorbidity with preterm birth are unavailable. We aimed to estimate the prevalence of term and preterm babies born small for gestational age (term-SGA and preterm-SGA), and the relation to low birthweight (<2500 g), in 138 countries of low and middle income in 2010. METHODS Small for gestational age was defined as lower than the 10th centile for fetal growth from the 1991 US national reference population. Data from 22 birth cohort studies (14 low-income and middle-income countries) and from the WHO Global Survey on Maternal and Perinatal Health (23 countries) were used to model the prevalence of term-SGA births. Prevalence of preterm-SGA infants was calculated from meta-analyses. FINDINGS In 2010, an estimated 32·4 million infants were born small for gestational age in low-income and middle-income countries (27% of livebirths), of whom 10·6 million infants were born at term and low birthweight. The prevalence of term-SGA babies ranged from 5·3% of livebirths in east Asia to 41·5% in south Asia, and the prevalence of preterm-SGA infants ranged from 1·2% in north Africa to 3·0% in southeast Asia. Of 18 million low-birthweight babies, 59% were term-SGA and 41% were preterm-SGA. Two-thirds of small-for-gestational-age infants were born in Asia (17·4 million in south Asia). Preterm-SGA babies totalled 2·8 million births in low-income and middle-income countries. Most small-for-gestational-age infants were born in India, Pakistan, Nigeria, and Bangladesh. INTERPRETATION The burden of small-for-gestational-age births is very high in countries of low and middle income and is concentrated in south Asia. Implementation of effective interventions for babies born too small or too soon is an urgent priority to increase survival and reduce disability, stunting, and non-communicable diseases. FUNDING Bill & Melinda Gates Foundation by a grant to the US Fund for UNICEF to support the activities of the Child Health Epidemiology Reference Group (CHERG). Dietary quality among men and women in 187 countries in 1990 and 2010: A systematic assessment Article Full-text available Fumiaki Imamura Renata Micha Shahab Khatibzadeh DrPH Dariush Mozaffarian Background: Healthy dietary patterns are a global priority to reduce non-communicable diseases. Yet neither worldwide patterns of diets nor their trends with time are well established. We aimed to characterise global changes (or trends) in dietary patterns nationally and regionally and to assess heterogeneity by age, sex, national income, and type of dietary pattern. Methods: In this systematic assessment, we evaluated global consumption of key dietary items (foods and nutrients) by region, nation, age, and sex in 1990 and 2010. Consumption data were evaluated from 325 surveys (71·7% nationally representative) covering 88·7% of the global adult population. Two types of dietary pattern were assessed: one reflecting greater consumption of ten healthy dietary items and the other based on lesser consumption of seven unhealthy dietary items. The mean intakes of each dietary factor were divided into quintiles, and each quintile was assigned an ordinal score, with higher scores being equivalent to healthier diets (range 0–100). The dietary patterns were assessed by hierarchical linear regression including country, age, sex, national income, and time as exploratory variables. Findings: From 1990 to 2010, diets based on healthy items improved globally (by 2·2 points, 95% uncertainty interval (UI) 0·9 to 3·5), whereas diets based on unhealthy items worsened (−2·5, −3·3 to −1·7). In 2010, the global mean scores were 44·0 (SD 10·5) for the healthy pattern and 52·1 (18·6) for the unhealthy pattern, with weak intercorrelation (r=–0·08) between countries. On average, better diets were seen in older adults compared with younger adults, and in women compared with men (p +1 Global migration and the changing distribution of sickle haemoglobin: A quantitative study of temporal trends between 1960 and 2000 Article Full-text available Frédéric B Piel Andrew Tatem Zhuojie Huang [...] David J Weatherall Background: Changes in the geographical distribution of genetic disorders are often thought to happen slowly, especially when compared with infectious diseases. Whereas mutations, genetic drift, and natural selection take place over many generations, epidemics can spread through large populations within a few days or weeks. Nevertheless, population movements can interfere with these processes, and few studies have been done of their eff ect on genetic disorders. We aimed to investigate the eff ect of global migration on the distribution of the sickle-cell gene-the most common and clinically significant haemoglobin structural variant. Methods: For each country, we extracted data from the World Bank’s Global Bilateral Migration Database about international human migrations between 1960 and 2000. We combined this information with evidence-based estimates of national HbS allele frequencies, generated within a Bayesian geostatistical framework, to analyse temporal changes in the net numbers of migrants, and classified countries with an index summarising these temporal trends. Findings: The number of international migrants increased from 92.6 million in 1960, to 165.2 million in 2000. The estimated global number of migrants with HbS increased from about 1.6 million in 1960, to 3.6 million in 2000. This increase was largely due to an increase in the number of migrants from countries with HbS allele frequencies higher than 10%, from 3.1 million in 1960, to 14.2 million in 2000. Additionally, the mean number of countries of origin for each destination country increased from 70 (SE 46) in 1960, to 98 (48) in 2000, showing an increasing diversity in the network of international migrations between countries. Our index of change map shows a patchy distribution of the magnitude of temporal changes, with the highest positive and negative values scattered across all continents. Interpretation: Global human population movements have had a substantial eff ect on the distribution of the HbS gene. Population movements can create a long-term burden on health-care systems. Our findings, which emphasise countries in which migration fluxes are changing the most, should increase awareness about the global burden of haemoglobinopathies and encourage policy makers to implement specific public health interventions, such as screening programmes and genetic counselling. Funding: Wellcome Trust, European Research Council, Bill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases-National Institutes of Health, the Research and Policy for Infectious Disease Dynamics program, Fogarty International Center. Global and regional burden of first-ever ischaemic and haemorrhagic stroke during 1990-2010: Findings from the Global Burden of Disease Study 2010 Literature Review Rita Krishnamurthi Valery Feigin Mohammad H Forouzanfar [...] Christopher Murray Background: The burden of ischaemic and haemorrhagic stroke varies between regions and over time. With differences in prognosis, prevalence of risk factors, and treatment strategies, knowledge of stroke pathological type is important for targeted region-specific health-care planning for stroke and could inform priorities for type-specific prevention strategies. We used data from the Global Burden of Diseases, Injuries, and Risk Factors Study 2010 (GBD 2010) to estimate the global and regional burden of first-ever ischaemic and haemorrhagic stroke during 1990–2010. Methods: We searched Medline, Embase, LILACS, Scopus, PubMed, Science Direct, Global Health Database, the WHO library, and regional databases from 1990 to 2012 to identify relevant studies published between 1990 and 2010. We applied the GBD 2010 analytical technique (DisMod-MR) to calculate regional and country-specific estimates for ischaemic and haemorrhagic stroke incidence, mortality, mortality-to-incidence ratio, and disability-adjusted life-years (DALYs) lost, by age group (aged Global, regional, and national trends in haemoglobin concentration and prevalence of total and severe anaemia in children and pregnant and non-pregnant women for 1995–2011: A systematic analysis of population-representative data Article Full-text available Gretchen A Stevens Mariel M Finucane Luz de Regil [...] Majid Ezzati Background: Low haemoglobin concentrations and anaemia are important risk factors for the health and development of women and children. We estimated trends in the distributions of haemoglobin concentration and in the prevalence of anaemia and severe anaemia in young children and pregnant and non-pregnant women between 1995 and 2011. Methods: We obtained data about haemoglobin and anaemia for children aged 6–59 months and women of childbearing age (15–49 years) from 257 population-representative data sources from 107 countries worldwide. We used health, nutrition, and household surveys; summary statistics from WHO's Vitamin and Mineral Nutrition Information System; and summary statistics reported by other national and international agencies. We used a Bayesian hierarchical mixture model to estimate haemoglobin distributions and systematically addressed missing data, non-linear time trends, and representativeness of data sources. We quantified the uncertainty of our estimates. Findings: Global mean haemoglobin improved slightly between 1995 and 2011, from 125 g/L (95% credibility interval 123–126) to 126 g/L (124–128) in non-pregnant women, from 112 g/L (111–113) to 114 g/L (112–116) in pregnant women, and from 109 g/L (107–111) to 111 g/L (110–113) in children. Anaemia prevalence decreased from 33% (29–37) to 29% (24–35) in non-pregnant women, from 43% (39–47) to 38% (34–43) in pregnant women, and from 47% (43–51) to 43% (38–47) in children. These prevalences translated to 496 million (409–595 million) non-pregnant women, 32 million (28–36 million) pregnant women, and 273 million (242–304 million) children with anaemia in 2011. In 2011, concentrations of mean haemoglobin were lowest and anaemia prevalence was highest in south Asia and central and west Africa. Interpretation: Children's and women's haemoglobin statuses improved in some regions where concentrations had been low in the 1990s, leading to a modest global increase in mean haemoglobin and a reduction in anaemia prevalence. Further improvements are needed in some regions, particularly south Asia and central and west Africa, to improve the health of women and children and achieve global targets for reducing anaemia. Funding: Bill & Melinda Gates Foundation, Grand Challenges Canada, and the UK Medical Research Council. Use of antiretroviral therapy in households and risk of HIV acquisition in rural KwaZulu-Natal, South Africa, 2004-12: A prospective cohort study Article Alain Vandormael Marie-Louise Newell Till Bärnighausen Frank Tanser Background: Studies of HIV-serodiscordant couples in stable sexual relationships have provided convincing evidence that antiretroviral therapy can prevent the transmission of HIV. We aimed to quantify the preventive effect of a public-sector HIV treatment and care programme based in a community with poor knowledge and disclosure of HIV status, frequent migration, late marriage, and multiple partnerships. Specifically, we assessed whether an individual's hazard of HIV acquisition was associated with antiretroviral therapy coverage among household members of the opposite sex. Methods: In this prospective cohort study, we linked patients' records from a public-sector HIV treatment programme in rural KwaZulu-Natal, South Africa, with population-based HIV surveillance data collected between 2004 and 2012. We used information about coresidence to construct estimates of HIV prevalence and antiretroviral therapy coverage for each household. We then regressed the time to HIV seroconversion for 14,505 individuals, who were HIV-uninfected at baseline and individually followed up over time regarding their HIV status, on opposite-sex household antiretroviral therapy coverage, controlling for household HIV prevalence and a range of other potential confounders. Findings: 2037 individual HIV seroconversions were recorded during 54,845 person-years of follow-up. For each increase of ten percentage points in opposite-sex household antiretroviral therapy coverage, the HIV acquisition hazard was reduced by 6% (95% CI 2–9), after controlling for other factors. This effect size translates into large reductions in HIV acquisition hazards when household antiretroviral therapy coverage is substantially increased. For example, an increase of 50 percentage points in household antiretroviral therapy coverage (eg, from 20% to 70%) reduced the hazard of HIV acquisition by 26% (95% CI 9–39). Interpretation: Our findings provide further evidence that antiretroviral therapy significantly reduces the risk of onward transmission of HIV in a real-world setting in sub-Saharan Africa. Awareness that antiretroviral therapy can prevent transmission to coresident sexual partners could be a powerful motivator for HIV testing and antiretroviral treatment uptake, retention, and adherence. Funding: Wellcome Trust and National Institute of Child Health and Human Development (US National Institutes of Health). 46 Citations Recommend Geographical and socioeconomic inequalities in women and children's nutritional status in Pakistan in 2011: An analysis of data from a nationally representative survey Full-text available Mariachiara Di Cesare Zaid Bhatti Sajid Bashir Soofi Zulfiqar Bhutta Pakistan has one of the highest levels of child and maternal undernutrition worldwide, but little information about geographical and socioeconomic inequalities is available. We aimed to analyse anthropometric indicators for childhood and maternal nutrition at a district level in Pakistan and assess the association of nutritional status with food security and maternal and household socioeconomic factors. We used data from the 2011 Pakistan National Nutrition Survey, which included anthropometric measurements for 33 638 children younger than 5 years and 24 826 women of childbearing age. We estimated the prevalences of stunting, wasting, and underweight among children and of underweight, overweight, and obesity in women for all 143 districts of Pakistan using a Bayesian spatial technique. We used a mixed-effect linear model to analyse the association of nutritional status with individual and household sociodemographic factors and food security. Stunting prevalence in Pakistan's districts ranged between 22% (95% credible interval 19-26) and 76% (69-83); the lowest figures for wasting and underweight were both less than 2·5% and the highest were 42% (34-50) for wasting and 54% (49-59) for underweight. In 106 districts, more women were overweight than were underweight; in 49 of these districts more women were obese than were underweight. Children were better nourished if their mothers were taller or had higher weight, if they lived in wealthier households, and if their mothers had 10 or more years of education. Severe food insecurity was associated with worse nutritional outcomes for both children and women. We noted large social and geographical inequalities in child and maternal nutrition in Pakistan, masked by national and provincial averages. Pakistan is also beginning to face the concurrent challenge of high burden of childhood undernutrition and overweight and obesity among women of reproductive age. Planning, implementation, and evaluation of programmes for food and nutrition should be based on district-level needs and outcomes. Bill & Melinda Gates Foundation, Grand Challenges Canada, UK Medical Research Council. Copyright © 2015 Di Cesare et al. Open Access article distributed under the terms of CC BY-NC-ND. Published by .. All rights reserved. Follow Share The WHO 2014 Global tuberculosis report-further to go Article Alimuddin Zumla Andrew George Virendra Sharma [...] Matt Oliver This article discusses how the 19th WHO global tuberculosis report 2014 provides an opportunity to think about the global tuberculosis strategy, and to assess just how much further effort is needed before global tuberculosis control can be achieved. Use of the Robson classification to assess cesarean section trends in 21 countries: A secondary analysis of two WHO multicountry surveys Article Full-text available Joshua Vogel Ana Pilar Betrán Nadia Vindevoghel [...] Rates of caesarean section surgery are rising worldwide, but the determinants of this increase, especially in low-income and middle-income countries, are controversial. In this study, we aimed to analyse the contribution of specific obstetric populations to changes in caesarean section rates, by using the Robson classification in two WHO multicountry surveys of deliveries in health-care facilities. The Robson system classifies all deliveries into one of ten groups on the basis of five parameters: obstetric history, onset of labour, fetal lie, number of neonates, and gestational age. Methods: We studied deliveries in 287 facilities in 21 countries that were included in both the WHO Global Survey of Maternal and Perinatal Health (WHOGS; 2004-08) and the WHO Multi-Country Survey of Maternal and Newborn Health (WHOMCS; 2010-11). We used the data from these surveys to establish the average annual percentage change (AAPC) in caesarean section rates per country. Countries were stratified according to Human Development Index (HDI) group (very high/high, medium, or low) and the Robson criteria were applied to both datasets. We report the relative size of each Robson group, the caesarean section rate in each Robson group, and the absolute and relative contributions made by each to the overall caesarean section rate. Findings: The caesarean section rate increased overall between the two surveys (from 26.4% in the WHOGS to 31.2% in the WHOMCS, p=0.003) and in all countries except Japan. Use of obstetric interventions (induction, prelabour caesarean section, and overall caesarean section) increased over time. Caesarean section rates increased across most Robson groups in all HDI categories. Use of induction and prelabour caesarean section increased in very high/high and low HDI countries, and the caesarean section rate after induction in multiparous women increased significantly across all HDI groups. The proportion of women who had previously had a caesarean section increased in moderate and low HDI countries, as did the caesarean section rate in these women. Interpretation: Use of the Robson criteria allows standardised comparisons of data across countries and timepoints and identifies the subpopulations driving changes in caesarean section rates. Women who have previously had a caesarean section are an increasingly important determinant of overall caesarean section rates in countries with a moderate or low HDI. Strategies to reduce the frequency of the procedure should include avoidance of medically unnecessary primary caesarean section. Improved case selection for induction and prelabour caesarean section could also reduce caesarean section rates. Training abroad? Not so difficult Marisa Dias Maria Orlova Mariana Pinto da Costa A solution pathway for preterm birth: Accelerating a priority research agenda Eve Lackritz Christopher B Wilson Alan E. Guttmacher Paul H Wise Effect of a comprehensive programme to provide universal access to care for sputum-smear-positive multidrug-resistant tuberculosis in China: A before-and-after study Renzhong Li Yunzhou Ruan Qiang Sun [...] Daniel P Chin China has a quarter of all patients with multidrug-resistant tuberculosis (MDRTB) worldwide, but less than 5% are in quality treatment programmes. In a before-and-after study we aimed to assess the effect of a comprehensive programme to provide universal access to diagnosis, treatment, and follow-up for MDRTB in four Chinese cities (population 18 million). We designated city-level hospitals in each city to diagnose and treat MDRTB. All patients with smear-positive pulmonary tuberculosis diagnosed in Center for Disease Control (CDC) clinics and hospitals were tested for MDRTB with molecular and conventional drug susceptibility tests. Patients were treated with a 24 month treatment package for MDRTB based on WHO guidelines. Outpatients were referred to the CDC for directly observed therapy. We capped total treatment package cost at US$4644. Insurance reimbursement and project subsidies limited patients' expenses to 10% of charges for services within the package. We compared data from a 12 month programme period (2011) to those from a retrospective survey of all patients with MDRTB diagnosed in the same cities during a baseline period (2006-09). 243 patients were diagnosed with MDRTB or rifampicin-resistant tuberculosis during the 12 month programme period compared with 92 patients (equivalent to 24 per year) during the baseline period. 172 (71%) of 243 individuals were enrolled in the programme. Time from specimen collection for resistance testing to treatment initiation decreased by 90% (from median 139 days [IQR 69-207] to 14 days [10-21]), the proportion of patients who started on appropriate drug regimen increased 2·7 times (from nine [35%] of 26 patients treated to 166 [97%] of 172), and follow-up by the CDC after initial hospitalisation increased 24 times (from one [4%] of 23 patients to 163 [99%] of 164 patients). 6 months after starting treatment, the proportion of patients remaining on treatment increased ten times (from two [8%] of 26 patients to 137 [80%] of 172), and 116 (67%) of 172 patients in the programme period had negative cultures or clinical-radiographic improvement. Patients' expenses for hospital admission after MDRTB diagnosis decreased by 78% (from $796 to $174), reducing the ratio of patients' expenses to annual household income from 17·6% to 3·5% (p<0·0001 for all comparisons between baseline and programme periods). However, 36 (15%) patients did not start or had to discontinue treatment in the programme period because of financial difficulties. This comprehensive programme substantially increased access to diagnosis, quality treatment, and affordable treatment for MDRTB. The programme could help China to achieve universal access to MDRTB care but greater financial risk protection for patients is needed. Bill & Melinda Gates Foundation. Copyright © 2015 Li et al. Open Access article distributed under the terms of CC BY-NC-ND. Published by .. All rights reserved. … Read more Global access to surgical care: A modelling study Mark Shrime More than 2 billion people are unable to receive surgical care based on operating theatre density alone. The vision of the Lancet Commission on Global Surgery is universal access to safe, affordable surgical and anaesthesia care when needed. We aimed to estimate the number of individuals worldwide without access to surgical services as defined by the Commission's vision. We modelled access to surgical services in 196 countries with respect to four dimensions: timeliness, surgical capacity, safety, and affordability. We built a chance tree for each country to model the probability of surgical access with respect to each dimension, and from this we constructed a statistical model to estimate the proportion of the population in each country that does not have access to surgical services. We accounted for uncertainty with one-way sensitivity analyses, multiple imputation for missing data, and probabilistic sensitivity analysis. At least 4·8 billion people (95% posterior credible interval 4·6-5·0 [67%, 64-70]) of the world's population do not have access to surgery. The proportion of the population without access varied widely when stratified by epidemiological region: greater than 95% of the population in south Asia and central, eastern, and western sub-Saharan Africa do not have access to care, whereas less than 5% of the population in Australasia, high-income North America, and western Europe lack access. Most of the world's population does not have access to surgical care, and access is inequitably distributed. The near absence of access in many low-income and middle-income countries represents a crisis, and as the global health community continues to support the advancement of universal health coverage, increasing access to surgical services will play a central role in ensuring health care for all. None. Copyright © 2015 Alkire et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved. 738 Reads 516 Citations A call for international accountability—preserving hope amid false protection Agnes Binagwaho Corine Karema Burden of disease in adults admitted to hospital in a rural region of coastal Kenya: An analysis of data from linked clinical and demographic surveillance systems Article Full-text available Anthony Etyang Kenneth Kabubei Munge Erick Bunyasi Estimates of the burden of disease in adults in sub-Saharan Africa largely rely on models of sparse data. We aimed to measure the burden of disease in adults living in a rural area of coastal Kenya with use of linked clinical and demographic surveillance data. We used data from 18 712 adults admitted to Kilifi District Hospital (Kilifi, Kenya) between Jan 1, 2007, and Dec 31, 2012, linked to 790 635 person-years of observation within the Kilifi Health and Demographic Surveillance System, to establish the rates and major causes of admission to hospital. These data were also used to model disease-specific disability-adjusted life-years lost in the population. We used geographical mapping software to calculate admission rates stratified by distance from the hospital. The main causes of admission to hospital in women living within 5 km of the hospital were infectious and parasitic diseases (303 per 100 000 person-years of observation), pregnancy-related disorders (239 per 100 000 person-years of observation), and circulatory illnesses (105 per 100 000 person-years of observation). Leading causes of hospital admission in men living within 5 km of the hospital were infectious and parasitic diseases (169 per 100 000 person-years of observation), injuries (135 per 100 000 person-years of observation), and digestive system disorders (112 per 100 000 person-years of observation). HIV-related diseases were the leading cause of disability-adjusted life-years lost (2050 per 100 000 person-years of observation), followed by non-communicable diseases (741 per 100 000 person-years of observation). For every 5 km increase in distance from the hospital, all-cause admission rates decreased by 11% (95% CI 7-14) in men and 20% (17-23) in women. The magnitude of this decline was highest for endocrine disorders in women (35%; 95% CI 22-46) and neoplasms in men (30%; 9-45). Adults in rural Kenya face a combined burden of infectious diseases, pregnancy-related disorders, cardiovascular illnesses, and injuries. Disease burden estimates based on hospital data are affected by distance from the hospital, and the amount of underestimation of disease burden differs by both disease and sex. The Wellcome Trust, GAVI Alliance. Health gains and financial risk protection afforded by public financing of selected interventions in Ethiopia: an extended cost-effectiveness analysis Joseph B Babigumira [...] The way in which a government chooses to finance a health intervention can affect the uptake of health interventions and consequently the extent of health gains. In addition to health gains, some policies such as public finance can insure against catastrophic health expenditures. We aimed to evaluate the health and financial risk protection benefits of selected interventions that could be publicly financed by the government of Ethiopia. We used extended cost-effectiveness analysis to assess the health gains (deaths averted) and financial risk protection afforded (cases of poverty averted) by a bundle of nine (among many other) interventions that the Government of Ethiopia aims to make universally available. These nine interventions were measles vaccination, rotavirus vaccination, pneumococcal conjugate vaccination, diarrhoea treatment, malaria treatment, pneumonia treatment, caesarean section surgery, hypertension treatment, and tuberculosis treatment. Our analysis shows that, per dollar spent by the Ethiopian Government, the interventions that avert the most deaths are measles vaccination (367 deaths averted per $100 000 spent), pneumococcal conjugate vaccination (170 deaths averted per $100 000 spent), and caesarean section surgery (141 deaths averted per $100 000 spent). The interventions that avert the most cases of poverty are caesarean section surgery (98 cases averted per $100 000 spent), tuberculosis treatment (96 cases averted per $100 000 spent), and hypertension treatment (84 cases averted per $100 000 spent). Our approach incorporates financial risk protection into the economic evaluation of health interventions and therefore provides information about the efficiency of attainment of both major objectives of a health system: improved health and financial risk protection. One intervention might rank higher on one or both metrics than another, which shows how intervention choice-the selection of a pathway to universal health coverage-might involve weighing up of sometimes competing objectives. This understanding can help policy makers to select interventions to target specific policy goals (ie, improved health or financial risk protection). It is especially relevant for the design and sequencing of universal health coverage to meet the needs of poor populations. Bill & Melinda Gates Foundation. Copyright © 2015 Verguet et al. Open access article published under the terms of CC BY-NC-SA. Published by .. All rights reserved. Cost-ffectiveness of two versus three or more doses of intermittent preventive treatment for malaria during pregnancy in sub-Saharan Africa: A modelling study of meta-analysis and cost data Silke Fernandes Elisa Sicuri Kassoum Kayentao [...] Kara Hanson In 2012, WHO changed its recommendation for intermittent preventive treatment of malaria during pregnancy (IPTp) from two doses to monthly doses of sulfadoxine-pyrimethamine during the second and third trimesters, but noted the importance of a cost-effectiveness analysis to lend support to the decision of policy makers. We therefore estimated the incremental cost-effectiveness of IPTp with three or more (IPTp-SP3+) versus two doses of sulfadoxine-pyrimethamine (IPTp-SP2). For this analysis, we used data from a 2013 meta-analysis of seven studies in sub-Saharan Africa. We developed a decision tree model with a lifetime horizon. We analysed the base case from a societal perspective. We did deterministic and probabilistic sensitivity analyses with appropriate parameter ranges and distributions for settings with low, moderate, and high background risk of low birthweight, and did a separate analysis for HIV-negative women. Parameters in the model were obtained for all countries included in the original meta-analysis. We did simulations in hypothetical cohorts of 1000 pregnant women receiving either IPTp-SP3+ or IPTp-SP2. We calculated disability-adjusted life-years (DALYs) for low birthweight, severe to moderate anaemia, and clinical malaria. We calculated cost estimates from data obtained in observational studies, exit surveys, and from public procurement databases. We give financial and economic costs in constant 2012 US$. The main outcome measure was the incremental cost per DALY averted. The delivery of IPTp-SP3+ to 1000 pregnant women averted 113·4 DALYs at an incremental cost of $825·67 producing an incremental cost-effectiveness ratio (ICER) of $7·28 per DALY averted. The results remained robust in the deterministic sensitivity analysis. In the probabilistic sensitivity analyses, the ICER was $7·7 per DALY averted for moderate risk of low birthweight, $19·4 per DALY averted for low risk, and $4·0 per DALY averted for high risk. The ICER for HIV-negative women was $6·2 per DALY averted. Our findings lend strong support to the WHO guidelines that recommend a monthly dose of IPTp-SP from the second trimester onwards. Malaria in Pregnancy Consortium and the Bill & Melinda Gates Foundation. Copyright © 2015 Fernandes et al. Open Access article distributed under the terms of CC BY-NC-SA. Published by .. All rights reserved. Incidence and severity of childhood pneumonia in the first year of life in a South African birth cohort: The Drakenstein Child Health Study Childhood pneumonia causes substantial mortality and morbidity. Accurate measurements of pneumonia incidence are scarce in low-income and middle-income countries, particularly after implementation of pneumococcal conjugate vaccine. We aimed to assess the incidence, severity, and risk factors for pneumonia in the first year of life in children enrolled in a South African birth cohort. This birth cohort study is being done at two sites in Paarl, a periurban area of South Africa. We enrolled pregnant women (>18 years) and followed up mother-infant pairs to 1 year of age. We obtained data for risk factors and respiratory symptoms. Children received 13-valent pneumococcal conjugate vaccine according to national immunisation schedules. We established pneumonia surveillance systems and documented episodes of ambulatory pneumonia and pneumonia warranting hospital admission. We calculated incidence rate ratios for pneumonia with mixed-effects Poisson regression. Between May 29, 2012 and May 31, 2014, we enrolled 697 infants who accrued 513 child-years of follow-up. We recorded 141 pneumonia episodes, with an incidence of 0·27 episodes per child-year (95% CI 0·23-0·32). 32 (23%) pneumonia cases were severe pneumonia, with an incidence of 0·06 episodes per child-year (95% CI 0·04-0·08). Two (1%) of 141 pneumonia episodes led to death from pneumonia. Maternal HIV, maternal smoking, male sex, and malnutrition were associated with an increased incidence of pneumonia. Pneumonia incidence was high in the first year of life, despite a strong immunisation programme including 13-valent pneumococcal conjugate vaccine. Incidence was associated with pneumonia risk factors that are amenable to interventions. Prevention of childhood pneumonia through public health interventions to address these risk factors should be strengthened. Bill & Melinda Gates Foundation, South African Thoracic Society, Federation of Infectious Diseases Societies of South Africa, and University of Cape Town. Copyright © 2015 le Roux et al. Open Access article distributed under the terms of CC BY-NC-ND. Published by .. All rights reserved. Download Recommend Health-care workers as agents of sustainable development Funding AIDS programmes in the era of shared responsibility: An analysis of domestic spending in 12 low-income and middle-income countries Stephen Resch Theresa Ryckman Robert Hecht As the incomes of many AIDS-burdened countries grow and donors' budgets for helping to fight the disease tighten, national governments and external funding partners increasingly face the following question: what is the capacity of countries that are highly affected by AIDS to finance their responses from domestic sources, and how might this affect the level of donor support? In this study, we attempt to answer this question. We propose metrics to estimate domestic AIDS financing, using methods related to national prioritisation of health spending, disease burden, and economic growth. We apply these metrics to 12 countries in sub-Saharan Africa with a high prevalence of HIV/AIDS, generating scenarios of possible future domestic expenditure. We compare the results with total AIDS financing requirements to calculate the size of the resulting funding gaps and implications for donors. Nearly all 12 countries studied fall short of the proposed expenditure benchmarks. If they met these benchmarks fully, domestic spending on AIDS would increase by 2·5 times, from US$2·1 billion to $5·1 billion annually, covering 64% of estimated future funding requirements and leaving a gap of around a third of the total $7·9 billion needed. Although upper-middle-income countries, such as Botswana, Namibia, and South Africa, would become financially self-reliant, lower-income countries, such as Mozambique and Ethiopia, would remain heavily dependent on donor funds. The proposed metrics could be useful to stimulate further analysis and discussion around domestic spending on AIDS and corresponding donor contributions, and to structure financial agreements between recipient country governments and donors. Coupled with improved resource tracking, such metrics could enhance transparency and accountability for efficient use of money and maximise the effect of available funding to prevent HIV infections and save lives. US Centers for Disease Control and Prevention. Copyright © 2015 Hecht et al. Open Access article distributed under the terms of CC BY-NC-ND. Published by .. All rights reserved. … Read more Prevalence of chronic obstructive pulmonary disease and associated risk factors in Uganda (FRESH AIR Uganda): A prospective cross-sectional observational study Bruce Kirenga Niels Chavannes [...] Thys van der Molen In sub-Saharan Africa, little is known about the damage to respiratory health caused by biomass smoke and tobacco smoke. We assessed the prevalence of chronic obstructive pulmonary disease (COPD) and related risk factors in a rural region of Uganda. We did this prospective observational cross-sectional study in rural Masindi, Uganda. We randomly selected people above the age of 30 years from 30 villages. Trained local health-care workers asked validated questionnaires and administered spirometry to participants. We defined COPD as FEV1:FVC less than the lower limit of normal. We calculated prevalence of COPD and tested its association with risk factors. Between April 13, and Aug 14, 2012, we invited 620 people to participate, of whom 588 provided acceptable spirometry and were analysed. Mean age was 45 years (SD 13·7); 297 (51%) were women. 546 (93%) were exposed to biomass smoke. The prevalence of COPD was 16·2% (15·4% in men, 16·8% in women). Prevalence was highest in people aged 30-39 years (17 [38%] of 45 men, 20 [40%] of 50 women). 20 (44%) of 45 men with COPD were current smokers (mean age 40 years, SD 7·5), 11 (24%) were former smokers (mean age 49 years, SD 11·0); four [8%] of 50 women were current smokers (mean age 52 years, SD 18·1), nine (18%) were former smokers (mean age 64 years, SD 16·2). Mean Clinical COPD Questionnaire score was 0·81 (SD 0·78), mean Medical Research Council dyspnoea score was 1·33 (SD 0·65); 28 (30%) of 95 patients had had one or more exacerbations past 12 months. COPD was associated with wheeze (odds ratio 2·17, 95% CI 1·09-4·34; p=0·028) and being a former smoker (1·96, 1·07-3·59; p=0·029). In this rural district of Uganda, COPD starts early in life. Major risk factors were biomass smoke for both sexes and tobacco smoke for men. In addition to high smoking prevalence in men, biomass smoke could be a major health threat to men and women in rural areas of Uganda. International Primary Care Respiratory Group. Copyright © 2015 van Gemert et al. Open Access article distributed under the terms of CC BY. Published by .. All rights reserved. UN Multi-Country Cross Sectional Study on Men and Violence. Prevalence of and factors associated with male perpetration of intimate partner violence: findings from the UN Multi-country Cross-sectional Study on Men and Violence in Asia and the Pacific Rachel Jewkes Emma Fulu Tim Roselli Claudia Garcia-Moreno Background: Rape perpetration is under-researched. In this study, we aimed to describe the prevalence of, and factors associated with, male perpetration of rape of non-partner women and of men, and the reasons for rape, from nine sites in Asia and the Pacific across six countries: Bangladesh, China, Cambodia, Indonesia, Papua New Guinea, and Sri Lanka. Methods: In this cross-sectional study, undertaken in January 2011–December 2012, for each site we chose a multistage representative sample of households and interviewed one man aged 18–49 years from each. Men self-completed questions about rape perpetration. We present multinomial regression models of factors associated with single and multiple perpetrator rape and multivariable logistic regression models of factors associated with perpetration of male rape with population-attributable fractions. Findings: We interviewed 10 178 men in our study (815–1812 per site). The prevalence of non-partner single perpetrator rape varied between 2·5% (28/1131; rural Bangladesh) and 26·6% (225/846; Bougainville, Papua New Guinea), multiple perpetrator rape between 1·4% (18/1246; urban Bangladesh) and 14·1% (119/846; Bougainville, Papua New Guinea), and male rape between 1·5% (13/880; Jayapura, Indonesia) and 7·7% (65/850; Bougainville, Papua New Guinea). 57·5% (587/1022) of men who raped a non-partner committed their first rape as teenagers. Frequent reasons for rape were sexual entitlement (666/909; 73·3%, 95% CI 70·3–76·0), seeking of entertainment (541/921; 58·7%, 55·0–62·4), and as a punishment (343/905; 37·9%, 34·5–41·4). Alcohol was a factor in 249 of 921 cases (27·0%, 95% CI 24·2–30·1). Associated factors included poverty, personal history of victimisation (especially in childhood), low empathy, alcohol misuse, masculinities emphasising heterosexual performance, dominance over women, and participation in gangs and related activities. Only 443 of 1933 men (22·9%, 95% CI 20·7–25·3) who had committed rape had ever been sent to prison for any period. Interpretation: Rape perpetration committed by men is quite frequent in the general population in the countries studied, as it is in other countries where similar research has been undertaken, such as South Africa. Prevention of rape is essential, and interventions must focus on childhood and adolescence, and address culturally rooted male gender socialisation and power relations, abuse in childhood, and poverty. Funding: Partners for Prevention—a UN Development Programme, UN Population Fund, UN Women, and UN Volunteers regional joint programme for gender-based violence prevention in Asia and the Pacific; UN Population Fund Bangladesh and China; UN Women Cambodia and Indonesia; United Nations Development Programme in Papua New Guinea and Pacific Centre; and the Governments of Australia, the UK, Norway, and Sweden. Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: A prospective birth cohort study from Brazil Cesar G Victora Bernardo Horta Christian Loret de Mola [...] Fernando C Barros Breastfeeding has clear short-term benefits, but its long-term consequences on human capital are yet to be established. We aimed to assess whether breastfeeding duration was associated with intelligence quotient (IQ), years of schooling, and income at the age of 30 years, in a setting where no strong social patterning of breastfeeding exists. A prospective, population-based birth cohort study of neonates was launched in 1982 in Pelotas, Brazil. Information about breastfeeding was recorded in early childhood. At 30 years of age, we studied the IQ (Wechsler Adult Intelligence Scale, 3rd version), educational attainment, and income of the participants. For the analyses, we used multiple linear regression with adjustment for ten confounding variables and the G-formula. From June 4, 2012, to Feb 28, 2013, of the 5914 neonates enrolled, information about IQ and breastfeeding duration was available for 3493 participants. In the crude and adjusted analyses, the durations of total breastfeeding and predominant breastfeeding (breastfeeding as the main form of nutrition with some other foods) were positively associated with IQ, educational attainment, and income. We identified dose-response associations with breastfeeding duration for IQ and educational attainment. In the confounder-adjusted analysis, participants who were breastfed for 12 months or more had higher IQ scores (difference of 3·76 points, 95% CI 2·20-5·33), more years of education (0·91 years, 0·42-1·40), and higher monthly incomes (341·0 Brazilian reals, 93·8-588·3) than did those who were breastfed for less than 1 month. The results of our mediation analysis suggested that IQ was responsible for 72% of the effect on income. Breastfeeding is associated with improved performance in intelligence tests 30 years later, and might have an important effect in real life, by increasing educational attainment and income in adulthood. Wellcome Trust, International Development Research Center (Canada), CNPq, FAPERGS, and the Brazilian Ministry of Health. Copyright © 2015 Victora et al. Open Access article distributed under the terms of CC BY. Published by .. All rights reserved. Think big, World Bank: Time for a public health safeguard Richard Seifman Sarah Kornblet Claire J Standley [...] Rebecca L Katz Moving beyond sanitation's diarrhoea fixation Adithya Pradyumna Prahlad Im Ganesh Ck Download Effectiveness of reactive oral cholera vaccination in rural Haiti: A case-control study and bias-indicator analysis Louise C Ivers Isabelle Johanne Hilaire Jessica E Teng [...] Molly F Franke Between April and June, 2012, a reactive cholera vaccination campaign was done in Haiti with an oral inactivated bivalent whole-cell vaccine. We aimed to assess the effectiveness of the vaccine in a case-control study and to assess the likelihood of bias in that study in a bias-indicator study. Residents of Bocozel or Grand Saline who were eligible for the vaccination campaign (ie, age ≥12 months, not pregnant, and living in the region at the time of the vaccine campaign) were included. In the primary case-control study, cases had acute watery diarrhoea, sought treatment at one of three participating cholera treatment units, and had a stool sample positive for cholera by culture. For each case, four control individuals who did not seek treatment for acute watery diarrhoea were matched by location of residence, enrolment time (within 2 weeks of the case), and age (1-4 years, 5-15 years, and >15 years). Cases in the bias-indicator study were individuals with acute watery diarrhoea with a negative stool sample for cholera. Controls were selected in the same manner as in the primary case-control study. Trained staff used standard laboratory procedures to do rapid tests and stool cultures from study cases. Participants were interviewed to collect data on sociodemographic characteristics, risk factors for cholera, and self-reported vaccination. Data were analysed by conditional logistic regression, adjusting for matching factors. From Oct 24, 2012, to March 9, 2014, 114 eligible individuals presented with acute watery diarrhoea and were enrolled, 25 of whom were subsequently excluded. 47 participants were analysed as cases in the vaccine effectiveness case-control study and 42 as cases in the bias-indicator study. 33 (70%) of 47 cholera cases self-reported vaccination versus 167 (89%) of 188 controls (vaccine effectiveness 63%, 95% CI 8-85). 27 (57%) of 47 cases had certified vaccination versus 147 (78%) of 188 controls (vaccine effectiveness 58%, 13-80). Neither self-reported nor verified vaccination was significantly associated with non-cholera diarrhoea (vaccine effectiveness 18%, 95% CI -208 to 78 by self-report and -21%, -238 to 57 by verified vaccination). Bivalent whole-cell oral cholera vaccine effectively protected against cholera in Haiti from 4 months to 24 months after vaccination. Vaccination is an important component of efforts to control cholera epidemics. National Institutes of Health, Delivering Oral Vaccines Effectively project, and Department of Global Health and Social Medicine at Harvard Medical School. Copyright © 2015 Ivers et al. Open Access article distributed under the terms of CC BY-NC-ND. Published by .. All rights reserved. Milinovich GJ, Magalhaes RJ, Hu W.Role of big data in the early detection of Ebola and other emerging infectious diseases. Lancet Glob Health 3:e20-e21 Gabriel J Milinovich Ricardo J Soares Magalhães Wenbiao Hu Building health research infrastructure in Rwanda Rwandan Research And Implementation Writing Group:Agnes Binagwaho Jeanine Condo Vedaste Ndahindwa [...] Edward J. Mills Download Recommend The neglected global burden of tuberculosis in pregnancy Matthew Adam Bates Peter Mwaba Point-of-care diagnostic tests for low-resource settings Patricia J Garcia Paul You Gina Fridley [...] Rosanna Peeling 99 Reads Preparedness of Tanzanian health facilities for outpatient primary care of hypertension and diabetes: A cross-sectional survey Robert N Peck Janneth Mghamba Fiona M Vanobberghen [...] Saidi Kapiga Historically, health facilities in sub-Saharan Africa have mainly managed acute, infectious diseases. Few data exist for the preparedness of African health facilities to handle the growing epidemic of chronic, non-communicable diseases (NCDs). We assessed the burden of NCDs in health facilities in northwestern Tanzania and investigated the strengths of the health system and areas for improvement with regard to primary care management of selected NCDs. Between November, 2012, and May, 2013, we undertook a cross-sectional survey of a representative sample of 24 public and not-for-profit health facilities in urban and rural Tanzania (four hospitals, eight health centres, and 12 dispensaries). We did structured interviews of facility managers, inspected resources, and administered self-completed questionnaires to 335 health-care workers. We focused on hypertension, diabetes, and HIV (for comparison). Our key study outcomes related to service provision, availability of guidelines and supplies, management and training systems, and preparedness of human resources. Of adult outpatient visits to hospitals, 58% were for chronic diseases compared with 20% at health centres, and 13% at dispensaries. In many facilities, guidelines, diagnostic equipment, and first-line drug therapy for the primary care of NCDs were inadequate, and management, training, and reporting systems were weak. Services for HIV accounted for most chronic disease visits and seemed stronger than did services for NCDs. Ten (42%) facilities had guidelines for HIV whereas three (13%) facilities did for NCDs. 261 (78%) health workers showed fair knowledge of HIV, whereas 198 (59%) did for hypertension and 187 (56%) did for diabetes. Generally, health systems were weaker in lower-level facilities. Front-line health-care workers (such as non-medical-doctor clinicians and nurses) did not have knowledge and experience of NCDs. For example, only 74 (49%) of 150 nurses had at least fair knowledge of diabetes care compared with 85 (57%) of 150 for hyptertension and 119 (79%) of 150 for HIV, and only 31 (21%) of 150 had seen more than five patients with diabetes in the past 3 months compared with 50 (33%) of 150 for hypertension and 111 (74%) of 150 for HIV. Most outpatient services for NCDs in Tanzania are provided at hospitals, despite present policies stating that health centres and dispensaries should provide such services. We identified crucial weaknesses (and strengths) in health systems that should be considered to improve primary care for NCDs in Africa and identified ways that HIV programmes could serve as a model and structural platform for these improvements. UK Medical Research Council. Share Orientia, Rickettsia, and Leptospira pathogens as causes of CNS infections in Laos: a prospective study Background: Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. Methods: Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fluid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. Findings: 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were significantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p Causes of non-malarial fever in Laos: A prospective study Background: Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods: For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings: With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p Viral CNS infections in children from a malaria-endemic area of Malawi: A prospective cohort study Mac Mallewa Background: Fever with reduced consciousness is an important cause of hospital admission of children in sub-Saharan Africa, with high mortality. Cerebral malaria, diagnosed when acute Plasmodium falciparum infection and coma are recorded with no other apparent reason, is one important cause. We investigated whether viruses could also be an important cause of CNS infection in such patients, and examined the relative contribution of viral pathogens and malaria parasitaemia. Methods: We did a prospective cohort study in Blantyre, Malawi. From March 1, 2002, to Aug 31, 2004, we enrolled children aged between 2 months and 15 years who were admitted to hospital with suspected non-bacterial CNS infections. Children with a cerebrospinal fluid (CSF) white cell count of less than 1000 cells per μL and negative bacterial microscopy and culture were deemed to have suspected viral CNS infection. Blood was examined for asexual forms of P falciparum. PCR was done on CSF or on post-mortem brain biopsy specimens to detect 15 viruses known to cause CNS infection. Findings: Full outcome data were available for 513 children with suspected viral CNS infection, of whom 94 (18%) died. 163 children (32%) had P falciparum parasitaemia, of whom 34 (21%) died. At least one virus was detected in the CNS in 133 children (26%), of whom 43 (33%) died. 12 different viruses were detected; adenovirus was the most common, affecting 42 children; mumps, human herpes virus 6, rabies, cytomegalovirus, herpes simplex virus 1, and enterovirus were also important. 45 (9%) of the 513 children had both parasitaemia and viral infection, including 27 (35%) of 78 diagnosed clinically with cerebral malaria. Children with dual infection were more likely to have seizures than were those with parasitaemia alone, viral infection only, or neither (p Urine lipoarabinomannan testing for diagnosis of pulmonary tuberculosis in children: A prospective study Lesley Workman [...] Urine tests for mycobacterial lipoarabinomannan might be useful for point-of-care diagnosis of tuberculosis in adults with advanced HIV infection, but have not been assessed in children. We assessed the accuracy of urine lipoarabinomannan testing for the diagnosis of pulmonary tuberculosis in HIV-positive and HIV-negative children. We prospectively recruited children (aged ≤15 years) who presented with suspected tuberculosis at a primary health-care clinic and paediatric referral hospital in South Africa, between March 1, 2009, and April 30, 2012. We assessed the diagnostic accuracy of urine lipoarabinomannan testing with lateral flow assay and ELISA, with mycobacterial culture of two induced sputum samples as the reference standard. Positive cultures were identified by acid-fast staining and tested to confirm Mycobacterium tuberculosis and establish susceptibility to rifampicin and isoniazid. 535 children (median age 42·5 months, IQR 19·1-66·3) had urine and two induced specimens available for testing. 89 (17%) had culture-confirmed tuberculosis and 106 (20%) had HIV. The lateral flow lipoarabinomannan test showed poor accuracy against the reference standard, with sensitivity of 48·3% (95% CI 37·6-59·2), specificity of 60·8% (56·1-65·3), and an area under the receiver operating characteristic curve of 0·53 (0·46-0·60) for children without HIV and 0·64 (0·51-0·76) for children with HIV. ELISA had poor sensitivity in children without HIV (sensitivity 3·0%, 95% CI 0·4-10·5) and children with HIV (0%, 0·0-14·3); overall specificity was 95·7% (93·4-97·4). Urine lipoarabinomannan tests have insufficient sensitivity and specificity to diagnose HIV-positive and HIV-negative children with tuberculosis and should not be used in this patient population. US National Institutes of Health, the National Health Laboratory Services Research Trust, the Medical Research Council of South Africa, and the Wellcome Trust. Children’s height and weight in rural and urban populations in low-income and middle-income countries: A systematic analysis of population-representative data Background: Urban living affects children's nutrition and growth, which are determinants of their survival, cognitive development, and lifelong health. Little is known about urban–rural differences in children's height and weight, and how these differences have changed over time. We aimed to investigate trends in children's height and weight in rural and urban settings in low-income and middle-income countries, and to assess changes in the urban–rural differentials in height and weight over time. Methods: We used comprehensive population-based data and a Bayesian hierarchical mixture model to estimate trends in children's height-for-age and weight-for-age Z scores by rural and urban place of residence, and changes in urban–rural differentials in height and weight Z scores, for 141 low-income and middle-income countries between 1985 and 2011. We also estimated the contribution of changes in rural and urban height and weight, and that of urbanisation, to the regional trends in these outcomes. Findings: Urban children are taller and heavier than their rural counterparts in almost all low-income and middle-income countries. The urban–rural differential is largest in Andean and central Latin America (eg, Peru, Honduras, Bolivia, and Guatemala); in some African countries such as Niger, Burundi, and Burkina Faso; and in Vietnam and China. It is smallest in southern and tropical Latin America (eg, Chile and Brazil). Urban children in China, Chile, and Jamaica are the tallest in low-income and middle-income countries, and children in rural areas of Burundi, Guatemala, and Niger the shortest, with the tallest and shortest more than 10 cm apart at age 5 years. The heaviest children live in cities in Georgia, Chile, and China, and the most underweight in rural areas of Timor-Leste, India, Niger, and Bangladesh. Between 1985 and 2011, the urban advantage in height fell in southern and tropical Latin America and south Asia, but changed little or not at all in most other regions. The urban–rural weight differential also decreased in southern and tropical Latin America, but increased in east and southeast Asia and worldwide, because weight gain of urban children outpaced that of rural children. Interpretation: Further improvement of child nutrition will require improved access to a stable and affordable food supply and health care for both rural and urban children, and closing of the the urban–rural gap in nutritional status. Funding: Bill & Melinda Gates Foundation, Grand Challenges Canada, UK Medical Research Council. … Read more The consequences of tobacco tax on household health and finances in rich and poor smokers in China: An extended cost-effectiveness analysis In China, there are more than 300 million male smokers. Tobacco taxation reduces smoking-related premature deaths and increases government revenues, but has been criticised for disproportionately affecting poorer people. We assess the distributional consequences (across different wealth quintiles) of a specific excise tax on cigarettes in China in terms of both financial and health outcomes. We use extended cost-effectiveness analysis methods to estimate, across income quintiles, the health benefits (years of life gained), the additional tax revenues raised, the net financial consequences for households, and the financial risk protection provided to households, that would be caused by a 50% increase in tobacco price through excise tax fully passed onto tobacco consumers. For our modelling analysis, we used plausible values for key parameters, including an average price elasticity of demand for tobacco of -0·38, which is assumed to vary from -0·64 in the poorest quintile to -0·12 in the richest, and we considered only the male population, which constitutes the overwhelming majority of smokers in China. Our modelling analysis showed that a 50% increase in tobacco price through excise tax would lead to 231 million years of life gained (95% uncertainty range 194-268 million) over 50 years (a third of which would be gained in the lowest income quintile), a gain of US$703 billion ($616-781 billion) of additional tax revenues from the excise tax (14% of which would come from the lowest income quintile, compared with 24% from the highest income quintile). The excise tax would increase overall household expenditures on tobacco by $376 billion ($232-505 billion), but decrease these expenditures by $21 billion (-$83 to $5 billion) in the lowest income quintile, and would reduce expenditures on tobacco-related disease by $24·0 billion ($17·3-26·3 billion, 28% of which would benefit the lowest income quintile). Finally, it would provide financial risk protection worth $1·8 billion ($1·2-2·3 billion), mainly concentrated (74%) in the lowest income quintile. Increased tobacco taxation can be a pro-poor policy instrument that brings substantial health and financial benefits to households in China. Bill & Melinda Gates Foundation and Dalla Lana School of Public Health. Copyright © 2015 Verguet et al. Open access article distributed under the terms of CC BY-NC-SA. Published by .. All rights reserved. 125 Citations Download Stroke risk factors in an incident population in urban and rural Tanzania: A prospective, community-based, case-control study Richard W Walker Ahmed Jusabani Eric Aris Ferdinand Mugusi The burden of stroke on health systems in low-income and middle-income countries is increasing. However, high-quality data for modifiable stroke risk factors in sub-Saharan Africa are scarce, with no community-based, case-control studies previously published. We aimed to identify risk factors for stroke in an incident population from rural and urban Tanzania. Stroke cases from urban Dar-es-Salaam and the rural Hai district were recruited in a wider study of stroke incidence between June 15, 2003, and June 15, 2006. We included cases with first-ever and recurrent stroke. Community-acquired controls recruited from the background census populations of the two study regions were matched with cases for age and sex and were interviewed and assessed. Data relating to medical and social history were recorded and blood samples taken. We included 200 stroke cases (69 from Dar-es-Salaam and 131 from Hai) and 398 controls (138 from Dar-es-Salaam and 260 from Hai). Risk factors were similar at both sites, with previous cardiac event (odds ratio [OR] 7·39, 95% CI 2·42-22·53; p<0·0001), HIV infection (5·61, 2·41-13·09; p<0·0001), a high ratio of total cholesterol to HDL cholesterol (4·54, 2·49-8·28; p<0·0001), smoking (2·72, 1·49-4·96; p=0·001), and hypertension (2·14, 1·09-4·17; p=0·026) identified as significant independent risk factors for stroke. In Hai, additional risk factors of diabetes (4·04, 1·29-12·64) and low HDL cholesterol (9·84, 4·06-23·84) were also significant. We have identified many of the risk factors for stroke already reported for other world regions. HIV status was an independent risk factor for stroke within an antiretroviral-naive population. Clinicians should be aware of the increased risk of stroke in people with HIV, even in the absence of antiretroviral treatment. The Wellcome Trust. Controlling Ebola through mHealth strategies James O'Donovan Amalia Bersin The current Ebola epidemic threatens to turn into one of the most devastating public health crises of modern times unless the international community responds in a coordinated, effective fashion. Owing to the high penetration of mobile phone subscriptions in Guinea (63/100 people), Liberia (60/100 people), and Sierra Leone (44/100 people),1 mHealth strategies could be a low-cost, high-impact solution to mapping outbreaks and providing education. Death and taxes Estimated need for surgery worldwide based on prevalence of diseases: A modelling strategy for the WHO Global Health Estimate Article Full-text available John Rose Thomas G Weiser Phil Hider Surgery is a foundational component of health-care systems. However, previous efforts to integrate surgical services into global health initiatives do not reflect the scope of surgical need and many health systems do not provide essential interventions. We estimate the minimum global volume of surgical need to address prevalent diseases in 21 epidemiological regions from the Global Burden of Disease Study 2010 (GBD). Prevalence data were obtained from GBD 2010 and organised into 119 disease states according to the WHO's Global Health Estimate (GHE). These data, representing 187 countries, were then apportioned into the 21 GBD epidemiological regions. Using previously defined values for the incident need for surgery for each of the 119 GHE disease states, we calculate minimum global need for surgery based on the prevalence of each condition in each region. We estimate that at least 321·5 million surgical procedures would be needed to address the burden of disease for a global population of 6·9 billion in 2010. Minimum rates of surgical need vary across regions, ranging from 3383 operations per 100 000 in central Latin America to 6495 operations per 100 000 in western sub-Saharan Africa. Global surgical need also varied across subcategories of disease, ranging from 131 412 procedures for nutritional deficiencies to 45·8 million procedures for unintentional injuries. The estimated need for surgical procedures worldwide is large and addresses a broad spectrum of disease states. Surgical need varies between regions of the world according to disease prevalence and many countries do not meet the basic needs of their populations. These estimates could be useful for policy makers, funders, and ministries of health as they consider how to incorporate surgical capacity into health systems. US National Institutes of Health. Copyright © 2015 Rose et al. Open access article published under the terms of CC BY-NC-ND. Published by Elsevier Ltd.. All rights reserved. An evolution in global health Keith Martin Zoe Mullan Richard Horton 4 Citations Download Effect of community-based voluntary counselling and testing on HIV incidence and social and behavioural outcomes (NIMH Project Accept; HPTN 043): A cluster-randomised trial Thomas J Coates David D Celentano Background: Although several interventions have shown reduced HIV incidence in clinical trials, the community-level effect of effective interventions on the epidemic when scaled up is unknown. We investigated whether a multicomponent, multilevel social and behavioural prevention strategy could reduce HIV incidence, increase HIV testing, reduce HIV risk behaviour, and change social and behavioural norms. Methods: For this phase 3 cluster-randomised controlled trial, 34 communities in four sites in Africa and 14 communities in Thailand were randomly allocated in matched pairs to receive 36 months of community-based voluntary counselling and testing for HIV (intervention group) or standard counselling and testing alone (control group) between January, 2001, and December, 2011. The intervention was designed to make testing more accessible in communities, engage communities through outreach, and provide support services after testing. Randomisation was done by a computer-generated code and was not masked. Data were collected at baseline (n=14 567) and after intervention (n=56 683) by cross-sectional random surveys of community residents aged 18–32 years. The primary outcome was HIV incidence and was estimated with a cross-sectional multi-assay algorithm and antiretroviral drug screening assay. Thailand was excluded from incidence analyses because of low HIV prevalence. This trial is registered at ClinicalTrials.gov, number NCT00203749. Findings: The estimated incidence of HIV in the intervention group was 1·52% versus 1·81% in the control group with an estimated reduction in HIV incidence of 13·9% (relative risk [RR] 0·86, 95% CI 0·73–1·02; p=0·082). HIV incidence was significantly reduced in women older than 24 years (RR=0·70, 0·54–0·90; p=0·0085), but not in other age or sex subgroups. Community-based voluntary counselling and testing increased testing rates by 25% overall (12–39; p=0·0003), by 45% (25–69; p Recommend Follow Effects of health-system strengthening on under-5, infant, and neonatal mortality: 11-year provincial-level time-series analyses in Mozambique Background: Knowledge of the relation between health-system factors and child mortality could help to inform health policy in low-income and middle-income countries. We aimed to quantify modifiable health-system factors and their relation with provincial-level heterogeneity in under-5, infant, and neonatal mortality over time in Mozambique. Methods: Using Demographic and Health Survey (2003 and 2011) and Multiple Indicator Cluster Survey (2008) data, we generated provincial-level time-series of child mortality in under-5 (ages 0–4 years), infant (younger than 1 year), and neonatal (younger than 1 month) age groups for 2000–10. We built negative binomial mixed models to examine health-system factors associated with changes in child mortality. Findings: Under-5 mortality rate was heterogeneous across provinces, with yearly decreases ranging from 11·1% (Nampula) to 1·9% (Maputo Province). Heterogeneity was greater for neonatal mortality rate, with only seven of 11 provinces showing significant yearly decreases, ranging from 13·6% (Nampula) to 4·2% (Zambezia). Health workforce density (adjusted rate ratio 0·94, 95% CI 0·90–0·98) and maternal and child health nurse density (0·96, 0·92–0·99) were both associated with reduced under-5 mortality rate, as were institutional birth coverage (0·94, 0·90–0·98) and government financing per head (0·80, 0·65–0·98). Higher population per health facility was associated with increased under-5 mortality rate (1·14, 1·02–1·28). Neonatal mortality rate was most strongly associated with institutional birth attendance, maternal and child nurse density, and overall health workforce density. Infant mortality rate was most strongly associated with institutional birth attendance and population per health facility. Interpretation: The large decreases in child mortality seen in Mozambique between 2000 and 2010 could have been partly caused by improvements in the public-sector health workforce, institutional birth coverage, and government health financing. Increased attention should be paid to service availability, because population per health facility is increasing across Mozambique and is associated with increased under-5 mortality. Investments in health information systems and new methods to track potentially increasing subnational health disparities are urgently needed. Funding: Doris Duke Charitable Foundation and Mozambican National Institute of Health. The drug and vaccine landscape for neglected diseases (2000-11): A systematic assessment Article Full-text available Belen Pedrique Nathalie Strub-Wourgaft Claudette Some Background: In 1975–99, only 1·1% of new therapeutic products had been developed for neglected diseases. Since then, several public and private initiatives have attempted to mitigate this imbalance. We analysed the research and development pipeline of drugs and vaccines for neglected diseases from 2000 to 2011. Methods: We searched databases of drug regulatory authorities, WHO, and clinical trial registries for entries made between Jan 1, 2000, and Dec 31, 2011. We defined neglected diseases as malaria, tuberculosis, diarrhoeal diseases, neglected tropical diseases (NTDs; WHO definition), and other diseases of poverty according to common definitions. Findings: Of the 850 new therapeutic products registered in 2000–11, 37 (4%) were indicated for neglected diseases, comprising 25 products with a new indication or formulation and eight vaccines or biological products. Only four new chemical entities were approved for neglected diseases (three for malaria, one for diarrhoeal disease), accounting for 1% of the 336 new chemical entities approved during the study period. Of 148 445 clinical trials registered in Dec 31, 2011, only 2016 (1%) were for neglected diseases. Interpretation: Our findings show a persistent insufficiency in drug and vaccine development for neglected diseases. Nevertheless, these and other data show a slight improvement during the past 12 years in new therapeutics development and registration. However, for many neglected diseases, new therapeutic products urgently need to be developed and delivered to improve control and potentially achieve elimination. Funding: None. Domestic and donor financing for tuberculosis care and control in low-income and middle-income countries: An analysis of trends, 2002-11, and requirements to meet 2015 targets Christopher Fitzpatrick Andrea Pantoja Background: Progress in tuberculosis control worldwide, including achievement of 2015 global targets, requires adequate financing sustained for many years. WHO began yearly monitoring of tuberculosis funding in 2002. We used data reported to WHO to analyse tuberculosis funding from governments and international donors (in real terms, constant 2011 US$) and associated progress in tuberculosis control in low-income and middle-income countries between 2002 and 2011. We then assessed funding needed to 2015 and how this funding could be mobilised. Methods: We included low-income and middle-income countries that reported data about financing for tuberculosis to WHO and had at least three observations between 2002 and 2011. When data were missing for specific country–year combinations, we imputed the missing data. We aggregated country-specific results for eight country groups defined according to income level, political and economic profile, geography, and tuberculosis burden. We compared absolute changes in total funding with those in the total number of patients successfully treated and did cross-country comparisons of cost per successfully treated patient relative to gross domestic product. We estimated funding needs for tuberculosis care and control for all low-income and middle-income countries to 2015, and compared these needs with domestic funding that could be mobilised. Findings: Total funding grew from $1·7 billion in 2002 to $4·4 billion in 2011. It was mostly spent on diagnosis and treatment of drug-susceptible tuberculosis. 43 million patients were successfully treated, usually for $100–500 per person in countries with high burdens of tuberculosis. Domestic funding rose from $1·5 billion to $3·9 billion per year, mostly in Brazil, Russia, India, China, and South Africa (BRICS), which collectively account for 45% of global cases, where national contributions accounted for more than 95% of yearly funding. Donor funding increased from $0·2 billion in 2002 to $0·5 billion in 2011, and accounted for a mean of 39% of funding in the 17 countries with the highest burdens (excluding BRICS) and a mean of 67% in low-income countries by 2011. BRICS and upper middle-income countries could mobilise almost all of their funding needs to 2015 from domestic sources. A full response to the tuberculosis epidemic to 2015, including investments to tackle multidrug-resistant tuberculosis, will require international donor funding of $1·6–2·3 billion each year. Interpretation: Funding for tuberculosis control increased substantially between 2002 and 2011, resulting in impressive and cost-effective gains. The increasing self-sufficiency of many countries, including BRICS, which account for almost half the world's tuberculosis cases, is a success story for control of tuberculosis. Nonetheless, international donor funding remains crucial in many countries and more is needed to achieve 2015 targets. Funding: None. Association between economic growth and early childhood undernutrition: Evidence from 121 Demographic and Health Surveys from 36 low-income and middle-income countries Malavika Subramanyam S V Subramanian Background: Economic growth is widely regarded as a necessary, and often sufficient, condition for the improvement of population health. We aimed to assess whether macroeconomic growth was associated with reductions in early childhood undernutrition in low-income and middle-income countries. Methods: We analysed data from 121 Demographic and Health Surveys from 36 countries done between Jan 1, 1990, and Dec 31, 2011. The sample consisted of nationally representative cross-sectional surveys of children aged 0–35 months, and the outcome variables were stunting, underweight, and wasting. The main independent variable was per-head gross domestic product (GDP) in constant prices and adjusted for purchasing power parity. We used logistic regression models to estimate the association between changes in per-head GDP and changes in child undernutrition outcomes. Models were adjusted for country fixed effects, survey-year fixed effects, clustering, and demographic and socioeconomic covariates for the child, mother, and household. Findings: Sample sizes were 462 854 for stunting, 485 152 for underweight, and 459 538 for wasting. Overall, 35·6% (95% CI 35·4–35·9) of young children were stunted (ranging from 8·7% [7·6–9·7] in Jordan to 51·1% [49·1–53·1] in Niger), 22·7% (22·5–22·9) were underweight (ranging from 1·8% [1·3–2·3] in Jordan to 41·7% [41·1–42·3] in India), and 12·8% (12·6–12·9) were wasted (ranging from 1·2% [0·6–1·8] in Peru to 28·8% [27·5–30·0] in Burkina Faso). At the country level, no association was seen between average changes in the prevalence of child undernutrition outcomes and average growth of per-head GDP. In models adjusted only for country and survey-year fixed effects, a 5% increase in per-head GDP was associated with an odds ratio (OR) of 0·993 (95% CI 0·989–0·995) for stunting, 0·986 (0·982–0·990) for underweight, and 0·984 (0·981–0·986) for wasting. ORs after adjustment for the full set of covariates were 0·996 (0·993–1·000) for stunting, 0·989 (0·985–0·992) for underweight, and 0·983 (0·979–0·986) for wasting. These findings were consistent across various subsamples and for alternative variable specifications. Notably, no association was seen between per-head GDP and undernutrition in young children from the poorest household wealth quintile. ORs for the poorest wealth quintile were 0·997 (0·990–1·004) for stunting, 0·999 (0·991–1·008) for underweight, and 0·991 (0·978–1·004) for wasting. Interpretation: A quantitatively very small to null association was seen between increases in per-head GDP and reductions in early childhood undernutrition, emphasising the need for direct health investments to improve the nutritional status of children in low-income and middle-income countries. Funding: None. Effect of preventive and curative interventions on hepatitis C virus transmission in Egypt (ANRS 1211): a modelling study Top-cited authors Jost B Jonas Universität Heidelberg 8169 Citations Rakhi Dandona Public Health Foundation of India Van C Lansingh Help Me See and IMO 7948 Citations Kovin Shunmugam Naidoo University of KwaZulu-Natal	https://www.researchgate.net/journal/The-Lancet-Global-Health-2214-109X
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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource275370CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2008-0825-FIs Part OfA related resource in which the described resource is physically or logically included.Box 1< a href="https://catalog.archives.gov/id/41027744" target="_blank" rel="noreferrer"> National Archives Catalog Description< /a>< a href="https://clinton.presidentiallibraries.us/items/show/48945"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20080825F-001-035-2016ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.4/27/2016https://clinton.presidentiallibraries.us/files/original/b7167dbc2c250cb0c6886b430bf91c51.pdf5cef2177e25cf40a05864127c122ad54Dublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceLippo GroupCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementOffice of CorrespondenceOffice of Counsel to the PresidentNational Security CouncilWhite House Operations OfficeOffice of Political AffairsOffice of Presidential PersonnelOffice of the Press Secretary (Press Office)Office of Public LiaisonWhite House Office of Records ManagementAutomated Records Management SystemNational Security CouncilDateA point or period of time associated with an event in the lifecycle of the resource1993-2000Is Part OfA related resource in which the described resource is physically or logically included.< strong>< a href="https://clinton.presidentiallibraries.us/items/show/48945"> Collection Finding Aid< /a>< /strong>IdentifierAn unambiguous reference to the resource within a given context2008-0825-FDescriptionAn account of the resourceThis collection consists of records dealing with the fundraising and campaign finance scandals associated with the Lippo Group and three Chinese businessmen—John Huang, Johnny Chung, and Yah Lin “Charlie” Trie. 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Clinton Presidential Library & MuseumExtentThe size or duration of the resource.517 folders in 29 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. 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The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.4/27/2016https://clinton.presidentiallibraries.us/files/original/dd7bd960b360d38536fb96aea5001c12.pdf7454538be7a7a3144ba826e6b7dc861aDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. 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The collection contains memorandums, emails, timelines, correspondence, lists, agendas, schedules, records of attendance, transcripts, and handwritten notes. The collection also includes materials produced for House and Senate investigations.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementClinton Presidential Records: White House Staff and Office FilesClinton Presidential Records: Automated Records Management SystemClinton Presidential Records: NSC Cable, Email, and Records Management SystemPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.517 folders in 29 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. 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The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.4/27/2016https://clinton.presidentiallibraries.us/files/original/96616ecec6ebb1c36b848cf0107de7cb.pdf4ae9de0ab710f6bf15a69ae10b04795dDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. 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The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.6/29/2017https://clinton.presidentiallibraries.us/files/original/ef6eda9563f36f0f0a1d5050077aab35.pdf53b2ca4bb27d8c16807d42698842c9d8Dublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291859CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239313"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-034-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.6/29/2017https://clinton.presidentiallibraries.us/files/original/415f2f162674d3aee4009d62486d9fcc.pdf9bc05631b94d5d9e1edad22935bc171dDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291668CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239311"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-033-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291666CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239309"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-032-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. 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Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291599CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239305"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-030-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumFormatThe file format, physical medium, or dimensions of the resourceAdobe Acrobat DocumentMediumThe material or physical carrier of the resource.Preservation-Reproduction-ReferenceDate CreatedDate of creation of the resource.6/29/2017https://clinton.presidentiallibraries.us/files/original/1cc7ade196c5e9dbfae9a41a71e7a3df.pdf9d6b9c160535ebd4379c45755ed3054aDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291598CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239303"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-029-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. 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For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resourceWHORM Subject File HUCreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementIs Part OfA related resource in which the described resource is physically or logically included.< a href="https://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>IdentifierAn unambiguous reference to the resource within a given context2012-0181-FDescriptionAn account of the resourceThe collection consists of records from WHORM (White House Office of Records Management) Subject File HU, Human Rights. The Subject File is a primary category containing information pertaining to human rights and civil rights, citizenship, segregation, ideologies and voting rights, and includes communications from the public expressing views about such matters. Subdivisions within the Subject File include genocide, civil disturbances, ideologies, equality for women, and equality in education and employment. There are also subdivisions for ethnic origin groups, such as Arab-American, Asian-American, African-American, Eastern European-American, Hungarian-American, Greek-American, Irish-American, and others. The records in this collection include memoranda, correspondence, reports, articles, and publications. Six diskettes were processed electronically and included in a single disk at the end of the collection.ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians made to the resource.Clinton Presidential Records: White House Office of Records ManagementPublisherAn entity responsible for making the resource availableWilliam J. Clinton Presidential Library & MuseumExtentThe size or duration of the resource.2,014 folders in 41 boxesTextA resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.Original FormatThe type of object, such as painting, sculpture, paper, photo, and additional dataPaperDublin CoreThe Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.TitleA name given to the resource291570CreatorAn entity primarily responsible for making the resourceWhite House Office of Records ManagementSubject FileHU013-20IdentifierAn unambiguous reference to the resource within a given context2012-0181-FIs Part OfA related resource in which the described resource is physically or logically included.Box 26< a href="https://catalog.archives.gov/id/122239301"> National Archives Catalog Description< /a>< a href="http://clinton.presidentiallibraries.us/items/show/57268"> Collection Finding Aid< /a>SourceA related resource from which the described resource is derived112767342-t-1127673-20120181F-026-028-2017ProvenanceA statement of any changes in ownership and custody of the resource since its creation that are significant for its authenticity, integrity, and interpretation. The statement may include a description of any changes successive custodians m	https://clinton.presidentiallibraries.us/items/browse?advanced%5B0%5D%5Belement_id%5D=39&advanced%5B0%5D%5Bterms%5D=HU013-20&advanced%5B0%5D%5Btype%5D=is+exactly&output=omeka-xml
Peoria, IL 1900 Federal Census ftp://ftp.us-census.org/pub/usgenweb/census/il/peoria/1900/ (File 2 of 3 for ED:89) This Census was transcribed by Lana Magiera and proofread by Amanda Hirsch for the USGenWeb Census Project http://www.us-census.org/ Copyright (c) 2003 by Lana Magiera <Transcriber@US-Census.org> ========================================================================== USGENWEB (US-CENSUS) NOTICE: These electronic pages may NOT be reproduced in any format for profit or presentation by any other organization. Non-commercial organizations desiring to use this material must obtain the consent of the transcriber prior to use. <Transcriber@US-Census.org> Individuals desiring to use this material in their own research may do so. ========================================================================== Formatted by USGenWeb Census Project File Manager, Connie Burkett All of the above information must remain when copied or downloaded. ========================================================================== Census Year 1900 Microfilm Roll #T623-333 State ILLINOIS County PEORIA ---------------------Begin Actual Transcription---------------------------------- =========================================================================================================================================================================================================================================================================================== CENSUS YEAR: 1900 STATE: IL COUNTY: Peoria TOWNSHIP: Peoria INCORPORATED-PLACE: Peoria City WARD-OF-CITY: First Ward Fifth Precinct MICROFILM#: T623-333 SUPV/DISTR: 8 ENUM/DISTR: 89 ENUMERATOR: Robert H. Kyle ENUM/DATE-RANGE: June 1 to 13 1900 =========================================================================================================================================================================================================================================================================================== BIRTH BIRTH S-M #YRS # #CHILD BIRTH FATHER MOTHER YEAR #YRS NATUR- MONTHS #MO AT CAN CAN SPEAK O-R O-F FARM #FARM PG# LN# ED SH# STREET HS# DW# FM# LAST NAME FIRST NAME RELATION RACE SEX MONTH YEAR AGE W-D MAR CHILD LIVING PLACE BIRTHPL BIRTHPL IMMI US ALIZED OCCUPATION UNEMPL SCHOOL READ WRITE ENG? HOME MORT HOME SCH (1) (2) (3) (3) (4) (5) (6) (7) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) =========================================================================================================================================================================================================================================================================================== 301B 51 89 4B New York Ave. 401 78 82 Burrows Martha J. Daughter W F May 1861 39 M 12 3 0 Illinois Massachusetts Ohio . . . . . . yes yes yes . . . . 301B 52 89 4B New York Ave. 401 78 82 Peters Wm. W. Son W M Oct 1872 27 S . . . Illinois Massachusetts Ohio . . . Hoseman (Fire dept.) 0 . yes yes yes . . . . 301B 53 89 4B New York Ave. 401 78 82 Peters Ellis H. Son W M June 1878 21 S . . . Illinois Massachusetts Ohio . . . Trav. Salesman 0 . yes yes yes . . . . 301B 54 89 4B New York Ave. 401 78 82 Johnson Mollie Boarder W F Feby 1882 18 S . . . Illinois Pennsylvania Iowa . . . . . . yes yes yes . . . . 301B 55 89 4B New York Ave. 323 79 83 Boylan John Head W M Jan 1841 59 M 20 . . Illinois Ireland Ireland . . . Farmer (retired) . . yes yes yes R . H . 301B 56 89 4B New York Ave. 323 79 83 Boylan Eva Wife W F Jan 1851 49 M 20 4 4 Illinois Germany Germany . . . . . . yes yes yes . . . . 301B 57 89 4B New York Ave. 323 79 83 Boylan Emmett Son W M Aug 1881 18 S . . . Illinois Illinois Illinois . . . At school . 3 yes yes yes . . . . 301B 58 89 4B New York Ave. 323 79 83 Boylan John B. Son W M June 1884 15 S . . . Illinois Illinois Illinois . . . At school . 9 yes yes yes . . . . 301B 59 89 4B New York Ave. 323 79 83 Boylan Louis Son W M July 1886 13 S . . . Illinois Illinois Illinois . . . At school . 9 yes yes yes . . . . 301B 60 89 4B New York Ave. 323 79 83 Boylan Archibald Son W M June 1888 11 S . . . Illinois Illinois Illinois . . . At school . 9 yes yes yes . . . . 301B 61 89 4B New York Ave. 323 79 83 Kendall Reuben Boarder W M June 1869 30 S . . . Illinois Illinois Illinois . . . Horse Buyer 0 . yes yes yes . . . . 301B 62 89 4B New York Ave. 323 79 83 Wiley Lemon H. Son in law W M Apr 1844 56 M 28 . . Pennsylvania Pennsylvania Pennsylvania . . . Musician 0 . yes yes yes . . . . 301B 63 89 4B New York Ave. 323 79 83 Wiley Alta Daughter W F May 1850 50 M 28 2 0 Michigan Massachusetts Massachusetts . . . . . . yes yes yes . . . . 301B 64 89 4B California Ave. 418 80 84 Fry Charles Head W M Mar 1826 72 M 10 . . England England England 1851 49 Na . . . yes yes yes O F H . 301B 65 89 4B California Ave. 418 80 84 Fry Hanna Wife W F May 1846 54 M 10 1 0 Canada (Eng) England England 1889 11 . . . . yes yes yes . . . . 301B 66 89 4B New York Ave. 315 81 85 O'Conner Hamilton Head W M Feby 1853 47 M 22 . . Illinois Ireland England . . . Clerk (R.R.) 0 . yes yes yes O M H . 301B 67 89 4B New York Ave. 315 81 85 O'Conner Mary Wife W F June 1855 45 M 22 8 8 Scotland Scotland Scotland 1872 28 . . . . yes yes yes . . . . 301B 68 89 4B New York Ave. 315 81 85 O'Conner Marian M. Daughter W F May 1879 21 S . . . Illinois Illinois Scotland . . . Stenographer (R.R.) 0 . yes yes yes . . . . 301B 69 89 4B New York Ave. 315 81 85 O'Conner Agnes E. Daughter W F May 1880 20 S . . . Illinois Illinois Scotland . . . . . . yes yes yes . . . . 301B 70 89 4B New York Ave. 315 81 85 O'Conner Mary F. Daughter W F Oct 1882 17 S . . . Illinois Illinois Scotland . . . At school . 9 yes yes yes . . . . 301B 71 89 4B New York Ave. 315 81 85 O'Conner John F. Son W M July 1885 14 S . . . Illinois Illinois Scotland . . . At school . 9 yes yes yes . . . . 301B 72 89 4B New York Ave. 315 81 85 O'Conner Grace E. Daughter W F Sept 1887 12 S . . . Illinois Illinois Scotland . . . At school . 9 yes yes yes . . . . 301B 73 89 4B New York Ave. 315 81 85 O'Conner Ethel Daughter W F Sept 1889 10 S . . . Illinois Illinois Scotland . . . At school . 9 yes yes yes . . . . 301B 74 89 4B New York Ave. 315 81 85 O'Conner Ruth M. Daughter W F Sept 1893 6 S . . . Illinois Illinois Scotland . . . At school . 9 . . . . . . . 301B 75 89 4B New York Ave. 315 81 85 O'Conner Dorothy E. Daughter W F April 1896 4 S . . . Illinois Illinois Scotland . . . . . . . . . . . . . 301B 76 89 4B New York Ave. 307 82 86 Walker Fred A. Head W M Aug 1864 35 M 12 . . Illinois Ohio Illinois . . . U.S. Mail Carrier 0 . yes yes yes R . H . 301B 77 89 4B New York Ave. 307 82 86 Walker Edna Wife W F Sept 1870 29 M 12 1 0 Illinois Indiana Ohio . . . . . . yes yes yes . . . . 301B 78 89 4B New York Ave. 307 82 86 Walker Allen P. Son W M Jany 1895 5 S . . . Illinois Illinois Illinois . . . . . . . . . . . . . 301B 79 89 4B New York Ave. 307 82 86 Smith Philip Father law W M May 1841 59 W . . . Germany Germany Germany 1856 44 Na Real estate 0 . yes yes yes . . . . 301B 80 89 4B New York Ave. 307 82 86 Walker Edward L. Brother W M July 1873 26 S . . . Illinois Ohio Illinois . . . Soldier enlisted (P.I.) 0 . yes yes yes . . . . 301B 81 89 4B New York Ave. 307 82 86 Lewis Cynthia A. Mother Law W F Oct 1849 50 W . 3 2 Ohio Ohio Ohio . . . . . . yes yes yes . . . . 301B 82 89 4B Illinois Ave. 509 83 87 Ledenhagen Ma** Head W M July 1870 30 M 2 . . Illinois Germany England . . . Salesman (butter) 0 . yes yes yes R . H . 301B 83 89 4B Illinois Ave. 509 83 87 Ledenhagen Alice E. Wife W F Dec 1872 27 M 2 0 0 Wisconsin Illinois England . . . . . . yes yes yes . . . . 301B 84 89 4B Illinois Ave. 503 84 88 Anderson Chas. C. Head W M Sept 1869 30 M 5 . . Nebraska Scotland Canada (Eng) . . . Passenger agent (R.R.) 0 . yes yes yes R . H . 301B 85 89 4B Illinois Ave. 503 84 88 Anderson Margaret J. Wife W F Jan 1871 29 M 5 1 1 Illinois Scotland Wales . . . . . . yes yes yes . . . . 301B 86 89 4B Illinois Ave. 503 84 88 Anderson Margaret R. Daughter W F Jan 1879 1 S . . . Illinois Nebraska Illinois . . . . . . . . . . . . . 301B 87 89 4B New York Ave. 400 85 89 Hailey Henry B. Head W M Oct 1856 45 M 23 . . Illinois Illinois Indiana . . . Insurance Agent 2 . yes yes yes O F H . 301B 88 89 4B New York Ave. 400 85 89 Hailey Ines Wife W F Oct 1859 40 M 23 4 3 Illinois Ohio Ohio . . . . . . yes yes yes . . . . 301B 89 89 4B New York Ave. 400 85 89 Stewart Claire A. Sister law W F June 1867 32 W . . . Illinois Ohio Ohio . . . . . . yes yes yes . . . . 301B 90 89 4B New York Ave. 400 85 89 Hailey Florence A. Daughter W F Jan 1897 3 S . . . Illinois Illinois Illinois . . . . . . . . . . . . . 301B 91 89 4B New York Ave. 400 85 89 Hunter Leota Niece W F Feby 1887 13 S . . . Illinois Illinois Illinois . . . At school . 9 yes yes yes . . . . 301B 92 89 4B New York Ave. 400 85 89 Hollingsworth William Son in law W M May 1878 22 M 0 . . Indiana Indiana Indiana . . . Insurance Agent 0 . yes yes yes . . . . 301B 93 89 4B New York Ave. 400 85 89 Hollingsworth P. A. Daughter W F Sept 1881 18 M 0 0 0 Illinois Illinois Illinois . . . . . . yes yes yes . . . . 301B 94 89 4B New York Ave. 400 85 89 Huff William Boarder W M Apr 1870 30 S . . . Illinois Illinois Illinois . . . . . . yes yes yes . . . . 301B 95 89 4B New York Ave. 402 86 90 Bury Patrick Head W M Oct 1860 39 M 15 . . Indiana Indiana Indiana . . . Steam Fitter 0 . yes yes yes O M H . 301B 96 89 4B New York Ave. 402 86 90 Bury Isabelle Wife W F Apr 1861 39 M 15 4 2 Michigan New York Canada (Eng) . . . . . . yes yes yes . . . . 301B 97 89 4B New York Ave. 402 86 90 Bury Harry W. Son W M July 1885 14 S . . . Kansas Indiana Michigan . . . At school . 9 yes yes yes . . . . 301B 98 89 4B New York Ave. 402 86 90 Bury Floyd Son W M Oct 1887 12 S . . . Illinois Indiana Michigan . . . At school . 9 yes yes yes . . . . 301B 99 89 4B New York Ave. 416 87 91 Gedney Lawrence Head W M Apr Unk Unk M . . . Illinois New York New York . . . . 0 . yes yes yes R . H . 301B 100 89 4B New York Ave. 416 87 91 Gedney Elizabeth Wife W F Unk Unk Unk M . . . Illinois New York New York . . . . . . . . . . . . . =========================================================================================================================================================================================================================================================================================== CENSUS YEAR: 1900 STATE: IL COUNTY: Peoria TOWNSHIP: Peoria INCORPORATED-PLACE: Peoria City WARD-OF-CITY: First Ward Fifth Precinct MICROFILM#: T623-333 SUPV/DISTR: 8 ENUM/DISTR: 89 ENUMERATOR: Robert H. Kyle ENUM/DATE-RANGE: June 1 to 13 1900 =========================================================================================================================================================================================================================================================================================== BIRTH BIRTH S-M #YRS # #CHILD BIRTH FATHER MOTHER YEAR #YRS NATUR- MONTHS #MO AT CAN CAN SPEAK O-R O-F FARM #FARM PG# LN# ED SH# STREET HS# DW# FM# LAST NAME FIRST NAME RELATION RACE SEX MONTH YEAR AGE W-D MAR CHILD LIVING PLACE BIRTHPL BIRTHPL IMMI US ALIZED OCCUPATION UNEMPL SCHOOL READ WRITE ENG? HOME MORT HOME SCH (1) (2) (3) (3) (4) (5) (6) (7) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) =========================================================================================================================================================================================================================================================================================== 302A 1 89 5A New York Ave. 416 87 91 Gedney Louis H. Son W M Nov 1872 27 S . . . Illinois New York New York . . . Butcher 0 . yes yes yes . . . . 302A 2 89 5A New York Ave. 416 87 91 Gedney Ernest Son W M Sept 1874 25 S . . . Illinois New York New York . . . Machinist 0 . yes yes yes . . . . 302A 3 89 5A New York Ave. 416 87 91 Gedney Otis Son W M July 1881 18 S . . . Illinois New York New York . . . Fitter (cycle factory) 0 . yes yes yes . . . . 302A 4 89 5A New York Ave. 414 88 92 Bohle Henry Head W M Feby 1866 34 M 4 . . Indiana Germany Germany . . . Car repairer (R.R.) 0 . yes yes yes O M H . 302A 5 89 5A New York Ave. 414 88 92 Bohle Emma Wife W F Feby 1868 32 M 4 0 0 Indiana Ohio Ohio . . . . . . yes yes yes . . . . 302A 6 89 5A New York Ave. 418 89 93 Larsen Mary A. Head W F Sept 1840 59 W . 3 2 Sweden Sweden Sweden 1867 33 . . . . yes yes yes O F H . 302A 7 89 5A New York Ave. 418 89 93 Larsen Frank W. Son W M Jan 1878 22 S . . . Illinois Sweden Sweden . . . Farm laborer 0 . yes yes yes . . . . 302A 8 89 5A New York Ave. 418 89 93 Larsen Ida M. Daughter W F May 1883 17 S . . . Illinois Sweden Sweden . . . . . . yes yes yes . . . . 302A 9 89 5A New York Ave. 420 90 94 Ryan John W. Head W M June 1847 52 M 32 . . Indiana Indiana Indiana . . . Piano tuner 0 . yes yes yes O F H . 302A 10 89 5A New York Ave. 420 90 94 Ryan Flora B. Wife W F Aug 1847 52 M 32 5 5 Illinois Vermont Vermont . . . . . . yes yes yes . . . . 302A 11 89 5A New York Ave. 420 90 94 Ryan Jessie W. Daughter W F Mar 1876 24 S . . . Illinois Indiana Illinois . . . Cashier (clothing store) 0 . yes yes yes . . . . 302A 12 89 5A New York Ave. 420 90 94 Ryan Mabel C. Daughter W F May 1878 22 S . . . Illinois Indiana Illinois . . . . . . yes yes yes . . . . 302A 13 89 5A New York Ave. 420 90 94 Ryan Lillian G. Daughter W F Apr 1883 17 S . . . Illinois Indiana Illinois . . . . . . yes yes yes . . . . 302A 14 89 5A New York Ave. 322 91 95 Walker Oliver P. Head W M Jan 1851 49 M 26 . . Illinois Indiana Indiana . . . C. Policeman 0 . yes yes yes O M H . 302A 15 89 5A New York Ave. 322 91 95 Walker Alice Wife W F Nov 1855 44 M 26 4 4 Illinois Virginia Illinois . . . . 0 . yes yes yes . . . . 302A 16 89 5A New York Ave. 322 91 95 Walker Charles W. Son W M Jan 1875 25 S . . . Illinois Illinois Illinois . . . Trav. Salesman 0 . yes yes yes . . . . 302A 17 89 5A New York Ave. 322 91 95 Walker Jessie E. Daughter W F July 1881 18 S . . . Illinois Illinois Illinois . . . Stenographer 0 . yes yes yes . . . . 302A 18 89 5A New York Ave. 322 91 95 Walker Oliver P. Son W M Aug 188 143 Woods Alice Boarder W F Dec 1894 5 S . . . Illinois Illinois Illinois . . . . . . . . . . . . . 304A 25 89 7A Glen Oak Ave. 1407 137 144 Henry Andrew Head W M Nov 1846 53 M 27 . . Ireland Ireland Ireland 1850 50 Na Insurance Agent 0 . yes yes yes O F H . 304A 26 89 7A Glen Oak Ave. 1407 137 144 Henry Julia R. Wife W F Feby 1850 50 M 27 5 5 Illinois Pennsylvania Pennsylvania . . . . . . yes yes yes . . . . 304A 27 89 7A Glen Oak Ave. 1407 137 144 Henry Harry G. Son W M Sept 1875 24 S . . . Illinois Ireland Illinois . . . Hotel Runner 0 . yes yes yes . . . . 304A 28 89 7A Glen Oak Ave. 1407 137 144 Henry Guy A. Son W M Mar 1878 22 S . . . Illinois Ireland Illinois . . . Optician 0 . yes yes yes . . . . 304A 29 89 7A Glen Oak Ave. 1407 137 144 Henry Mary E. Daughter W F Jan 1880 20 S . . . Illinois Ireland Illinois . . . At school . 9 yes yes yes . . . . 304A 30 89 7A Glen Oak Ave. 1407 137 144 Henry Bruce A. Son W M May 1884 16 S . . . Illinois Ireland Illinois . . . At school . 9 yes yes yes . . . . 304A 31 89 7A Glen Oak Ave. 1407 137 144 Henry Laura Daughter W F Sept 1886 13 S . . . Illinois Ireland Illinois . . . At school . 9 yes yes yes . . . . 304A 32 89 7A Glen Oak Ave. 1407 137 144 Odell Jay Nephew W M Nov 1888 11 S . . . Nebraska Illinois Illinois . . . At school . 9 yes yes yes . . . . 304A 33 89 7A Glen Oak Ave. 1409 138 145 Barfield William Head W M Sept 1818 81 M 55 . . Mississippi Georgia So. Carolina . . . . . . yes yes yes O F H . 304A 34 89 7A Glen Oak Ave. 1409 138 145 Barfield Margaret C. Wife W F Oct 1823 76 M 55 7 7 Ohio New Jersey New Jersey . . . . . . yes yes yes . . . . 304A 35 89 7A Glen Oak Ave. 1501 139 146 Behrends John Head W M May 1836 64 M 33 . . Germany Germany Germany 1872 28 . Ice Dealer 0 . yes yes yes O F H . 304A 36 89 7A Glen Oak Ave. 1501 139 146 Behrends Anna J. Wife W F May 1839 61 M 33 1 0 Germany Germany Germany 1872 28 . . . . yes yes yes . . . . 304A 37 89 7A Glen Oak Ave. 1501 139 146 Coal Anna Servant W F Apr 1882 18 S . . . Germany Germany Germany 1885 15 . Servant 0 . yes yes yes . . . . 304A 38 89 7A Glen Oak Ave. 1505 140 147 Wyant Fredrick Head W M May 1865 35 M 5 . . Kentucky Kentucky Kentucky . . . Baker 0 . yes yes yes R . H . 304A 39 89 7A Glen Oak Ave. 1505 140 147 Wyant Emma L. Wife W F Apr 1872 28 M 5 2 2 Wisconsin Germany Germany . . . . . . yes yes yes . . . . 304A 40 89 7A Glen Oak Ave. 1505 140 147 Wyant Edna A. Daughter W F June 1897 2 S . . . Illinois Kentucky Wisconsin . . . . . . . . . . . . . 304A 41 89 7A Glen Oak Ave. 1505 140 147 Wyant Fred, Jr. Son W M Nov 1899 6/12 S . . . Illinois Kentucky Wisconsin . . . . . . . . . . . . . 304A 42 89 7A Glen Oak Ave. 1509 141 148 Lester John Head W M Apr 1868 32 M . . . Iowa Iowa Iowa . . . Baggage Man 0 . yes yes yes R . H . 304A 43 89 7A Glen Oak Ave. 1509 141 148 Lester Ada Wife W F Mar 1867 33 M . . . Illinois Germany Kentucky . . . . . . yes yes yes . . . . 304A 44 89 7A Glen Oak Ave. 1509 141 148 Lester Hester May Daughter W F Jan 1891 8 S . . . Iowa Iowa Illinois . . . At school . 9 . . . . . . . 304A 45 89 7A Glen Oak Ave. 1509 141 148 Lester William F. Son W M Oct 1892 7 S . . . Iowa Iowa Illinois . . . At school . 9 . . . . . . . 304A 46 89 7A Glen Oak Ave. 1509 141 148 Lester Bertha B. Daughter W F Feby 1898 2 S . . . Iowa Iowa Illinois . . . . . . . . . . . . . 304A 47 89 7A E. Nebraska Ave. 810 142 149 Cunningham John L. Head W M Mar 1859 41 M . . . Pennsylvania Pennsylvania Pennsylvania . . . Trav. Salesman 0 . yes yes yes R . H . 304A 48 89 7A E. Nebraska Ave. 810 142 149 Cunningham Frances M. Wife W F Mar 1868 32 M . . . Illinois Illinois Ohio . . . . . . yes yes yes . . . . 304A 49 89 7A E. Nebraska Ave. 810 142 149 Cunningham Hazel Daughter W F Aug 1883 16 S . . . Illinois Pennsylvania Pennsylvania . . . At school . 9 yes yes yes . . . . 304A 50 89 7A E. Nebraska Ave. 810 142 149 Smith Anna L. Servant W F Apr 1880 20 S . . . Ohio Ohio Ohio . . . Servant . . yes yes yes . . . .	http://www.us-census.org/pub/usgenweb/census/il/peoria/1900/ed089-pg301b.txt
Agriculture \| Free Full-Text \| The Initiation of a Phytosociological Study on Certain Types of Medicinal Plants The cultivation of medicinal plants represents great necessity and topicality these days, given that the pharmaceutical industry requires high quality raw materials in large quantities. Those are used for the production of food supplements/phytomedicines/medical devices or gemmo-derivatives’ products. Starting from these premises, this present study aimed to culture common batches of different associations of medicinal plants in order to quantify the fabrication of plant products, but also to observe possible changes in their internal structure, in direct correlation with the biosynthesis of active principles. The crops were monitored in 2018–2021. It was found that in all the common crops compared to the control ones, the amount of vegetable product provided was much higher (for example, the thyme-rosemary crop produced 730 g of fresh vegetable plants, compared with 540 g in the control crop; St. John’s Wort in culture with lemon balm delivered 1934 g of vegetable product, compared with 1423 g obtained from the control crop; mint was grown with lemon balm and produced a double amount of vegetable mass compared with the control crop). The presence of numerous glandular hairs in the samples from the phytosociological groups for the species from the Lamiaceae family, could explain the difference in the volatile oil content (4 mL/100 g produced by rosemary from the thyme-rosemary crop compared with 3.6 mL/100 g from the control one; 6.6 mL/100 g generated by thyme from the thyme-rosemary crop compared with 3.6 mL/100 from the control group; 2 mL/100 g of lemon balm volatile oil from the mint-lemon balm compared with 0.6 mL/100 g). The content of other types of active principles is dependent on the culture association. From results analysis it was found that in the phytosociological groups, flavones, PCAs and total polyphenols were significantly higher compared to control ones (2.4413 ± 0.1858 g flavones expressed in rutin/100 g in the thyme dried leaves from thyme-rosemary to 1.9317 ± 0.0947 g flavones produced by the control thyme; 9.9461 ± 0.8385 g PCAs expressed in chlorogenic acid/100 g for the same sample compared with 6.9709 ± 1.4921 g produced by the control batch; 11.1911 ± 0.7959 g TPC expressed in tannic acid/100 g in the thyme dried leaves from the thyme-rosemary phytosociological crop to 6.0393 ± 0.3204 g from the control one). The obtained results can be a starting point regarding the potential associations of medicinal plants in crops, in order to obtain a qualitative and quantitative vegetal mass. The Initiation of a Phytosociological Study on Certain Types of Medicinal Plants by Emanuela Alice Luță 1,* , Manuela Ghica 2,* and Cerasela Elena Gîrd 1 Department of Pharmacognosy, Phytochemistry and Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy “Carol Davila”, 6 Traian Vuia Street, 020956 Bucharest, Romania * Authors to whom correspondence should be addressed. Agriculture 2022 , 12 (2), 283; https://doi.org/10.3390/agriculture12020283 Received: 27 December 2021 / Revised: 11 February 2022 / Accepted: 13 February 2022 / Published: 16 February 2022 (This article belongs to the Topic Grand Challenges of Advanced Technologies in Sustainable Agriculture 4.0: Future Farming, Harvesting and Preservation ) Abstract : The cultivation of medicinal plants represents great necessity and topicality these days, given that the pharmaceutical industry requires high quality raw materials in large quantities. Those are used for the production of food supplements/phytomedicines/medical devices or gemmo-derivatives’ products. Starting from these premises, this present study aimed to culture common batches of different associations of medicinal plants in order to quantify the fabrication of plant products, but also to observe possible changes in their internal structure, in direct correlation with the biosynthesis of active principles. The crops were monitored in 2018–2021. It was found that in all the common crops compared to the control ones, the amount of vegetable product provided was much higher (for example, the thyme-rosemary crop produced 730 g of fresh vegetable plants, compared with 540 g in the control crop; St. John’s Wort in culture with lemon balm delivered 1934 g of vegetable product, compared with 1423 g obtained from the control crop; mint was grown with lemon balm and produced a double amount of vegetable mass compared with the control crop). The presence of numerous glandular hairs in the samples from the phytosociological groups for the species from the Lamiaceae family, could explain the difference in the volatile oil content (4 mL/100 g produced by rosemary from the thyme-rosemary crop compared with 3.6 mL/100 g from the control one; 6.6 mL/100 g generated by thyme from the thyme-rosemary crop compared with 3.6 mL/100 from the control group; 2 mL/100 g of lemon balm volatile oil from the mint-lemon balm compared with 0.6 mL/100 g). The content of other types of active principles is dependent on the culture association. From results analysis it was found that in the phytosociological groups, flavones, PCAs and total polyphenols were significantly higher compared to control ones (2.4413 ± 0.1858 g flavones expressed in rutin/100 g in the thyme dried leaves from thyme-rosemary to 1.9317 ± 0.0947 g flavones produced by the control thyme; 9.9461 ± 0.8385 g PCAs expressed in chlorogenic acid/100 g for the same sample compared with 6.9709 ± 1.4921 g produced by the control batch; 11.1911 ± 0.7959 g TPC expressed in tannic acid/100 g in the thyme dried leaves from the thyme-rosemary phytosociological crop to 6.0393 ± 0.3204 g from the control one). The obtained results can be a starting point regarding the potential associations of medicinal plants in crops, in order to obtain a qualitative and quantitative vegetal mass. Keywords: anatomical characteristics ; phytosociology ; raw vegetable materials ; vegetation monitoring 1. Introduction Science in the plant biology field is in continuous progress, and phytosociology, a branch of phytogeography, occupies an important place in research, in order to use plant communities as environmental indicators. By analogy with plant taxonomy, phytosociological classification (syntaxonomy) places vegetation units in a hierarchical system, based on varying degrees of floristic resemblance [ 1 ]. Nowadays, this is the main method used throughout Europe and is also applied in North Asia and in different regions of Africa and Latin America [ 1 ]. While its application in North America has remained limited, recently it has launched the USNVC-US (National Vegetation Classification), which recognizes the importance of consistent hierarchical classification systems and adopts ideas from the Braun–Blanquet approach in a modified terminology [ 2 ]. While traditional phytosociologists believed that it can represent a whole set (portion) of vegetation with a single “typical” relevance, current phytosociology is seen as a statistical approach that aims to characterize vegetation types through combined information from several different plots [ 3 ]. The usefulness of molecular phylogeny and phylogenomics in speculating chemodiversity and bioprospecting is also highlighted within the context of natural-product-based drug discovery and development [ 4 ]. To understand the notion of a plant community, it has to be kept in mind that plant species that are components of a plant community grow together in a given area because they have similar requirements for existence [ 5 ] in terms of environmental factors, such as: light, temperature, humidity, nutrients, etc. [ 6 ]. Plant association can be defined as a group of plant species that grow together in a certain area and have a mutual alliance or affinity of a certain type between them. Following this relationship, it will be possible to observe, initially macroscopically and later microscopically, if this process will lead to changes or new acquired active principles [ 7 ]. The use of herbs in the treatment of certain diseases has been of high interest in recent decades. Different types of human communities traditionally use such herbs [ 8 ]. Some forest regions are extremely rich in their composition, and people then harvest them in excess, leading to the extinction of the species or even a change in the entire vegetation. A study was conducted on the natural medicinal plants in the Chalsa forest chain, located at the foot of the Himalayas, and sounded the alarm for a thorough phytosociological investigation, including an allelopathic analysis and an analysis of the soil seed bank [ 9 ]. In the study of Hatami E. et al. (2019), the importance of the symbiosis between plants and fungi was pointed out in the regeneration of soils contaminated involuntarily and/or voluntarily with oil residues [ 10 ]. Phytoremediation of lead-contaminated soils presented in the study organized by Saleem M et al. (2018), talks about the association of rhizobacteria in order to increase the resistance of plants to these types of contaminated soils [ 11 ]. A study conducted on five natural populations of Terminalia chebula Retz. realized by Singh S. et colb. (2019) aimed to analyze the structure of the vegetation and the distribution pattern of different species of trees and shrubs in these populations. It was able to provide information on the abundance, distribution, and rate of change in species composition [ 12 ]. Maharani D. et al. (2022) found that it is possible to optimize the land in the forest subsoil with the help of tubers that are resistant to shade, which can provide medium-term benefit for both forestry and food for the local population [ 13 ]. Plants that grow together have a common relationship with each other and with nature. The number of species sheltered in a community is an important factor from the ecological point of view since it seems to increase as the community becomes more stable. Those species accommodated in a plantation area were found in a higher sum compared with a degraded area [ 14 ]. According to studies by Jamil N. et al. (2022) and Koskey G. et al. (2022), one of the ways to increase the sustainability of some crops would be the reintroduction of intercropping crops; one of the main factors influencing the development of plants in the crop is the soil and climatic conditions, which can be translated into the horizontal or vertical evolution of those crops [ 15 , 16 ]. The interactions between certain types of plants and the environment have led to different types of vegetation occurring in different areas. The forests in Kandi Siwaliks, India, have witnessed the reduction of forested areas, isolation of smaller patches, habitat loss and a rise in disturbance level over the years. Therefore, to conserve plant diversity around the forest–village/town interfaces a protective buffer of edge species around newly created fragmented forest patches is required to protect the core species. The abundance to frequency ratio indicated that most of the species of shrub-sapling, herb-seedling and trees were contagiously distributed except a few species of trees, which showed random distribution pattern. The massive deforestation found in different parts of the globe has shown a much lower floristic development of plant species that coexisted with different species of trees. The regeneration of the soils, but also of the green space specific to the forests, is dependent on the coexistence in the natural environment of these plant–tree associations, a fact observed in the subtropical forests, especially in the Asian area [ 17 , 18 , 19 ]. The study conducted by Li L. et al. (2022) on the intercropping of different plant species aimed at increasing the amount of biomass produced and as much as possible reducing the supplementation of soil with organic fertilizers. The adaptation of crops in intercalated zones as well as the production of a large amount of plant biomass can be explained by the significant increase in soil microbial diversity in these types of cultures, compared to their reduction in chemical crops [ 20 ]. The aim of this study is to generate compatible batches of medicinal plants that can be grown together in order to produce much higher plant mass, while also assuming that the content of secondary metabolites may be higher. Based on this premise, the objectives of the study were: the establishment of medicinal plant cultures in common crops and observation of their development over a longer period of time along with comparation to control crops. The plant mass production and the content of secondary metabolites (volatile oils, flavones, PCAs, and total polyphenols content) were also analyzed. In these plants, the medicinal association type of family and/or the therapeutic component were taken into account. 2. Materials and Methods In this phytosociological study vegetable raw material with similar active principles have been selected, but belonging to different species or families. In order to carry out the scientific research, it was necessary to draw up a structured plan in stages conducted over several years, including both theoretical and practical parts. The following types of medicinal plants were associated in cultures based on therapeutic effects: crop 1— Mentha × piperita L. and Melissa officinalis L. (aromatic medicinal plants from the Lamiaceae family); crop 2— Thymus vulgaris L. and Calendula officinalis L. (associated in gastrointestinal diseases); crop 3— Rosmarinus officinalis L. and Matricaria chamomilla L. (source of volatile oil); crop 4— Hypericum perforatum L. and Chelidonium majus L. (associated in hepato-biliary disorders). Each phytosociological crop was accompanied by the corresponding control crop. 2.1. Description of the Study Site and Cultivation Condition All of the medicinal plants were pre-planted in experimental crops with the size of 50 cm × 300 cm, and 400 cm intervals between batches. The distance between the planted seedlings was established depending on the height of the young plants that were planted >30 cm. Transplanting was performed at a depth of 25 cm and 5 seedlings/batch were used. The scientific experiment was carried out in the suburban area of Turnu Măgurele, Teleorman County, Romania (43°44′44.16″ N, 24°52′53.40″ E), starting in 2018. The average annual temperature in Turnu Măgurele is 11.5 °C, the average in the warm months is 23 °C, and the average in the cold months drops below −2 °C. It is characterized by a high caloric potential, high amplitudes of air temperature, low amounts of precipitation and often in a torrential regime in summer, and frequent periods of drought [ 21 ]. The crops were observed in 2018, and were then analyzed in the following years, including 2021. A series of techno-agricultural works were carried out (land preparation-shredding, leveling and irrigation, weed control, seedling production, replanting), as seen in Figure 1 . 2.2. Experimental Procedures The phytosociological crops as well as the control ones were monitored during their development (2018–2021) by the horticultural engineer Ciocăneală Ștefan, being subjected to the same operations such as watering, weeding, etc. Seedlings were dug from chemically untreated seeds, using prolonged germination to improve the quality of the active ingredients [ 22 ]. The replanting of seedlings with new medicinal plants, as well as the completion of existing ones, was done every year in April. The control crops were planted at a distance from the culture, in order not to be influenced. Each medicinal plant from the phytosociological crop had a control one consisting of the same number of seedlings. During the maturation of the harvest, the morphological and phytochemical aspects were examined by comparing every crop with the control one. The research also sought to highlight potential morphological and anatomical changes in freshly harvested plant products using a stereomicroscope (ZEISS Stemi 508 Greenough Stereo Microscope with 8:1 Zoom, details up to 50× magnification). 2.3. Dosing Active Chemical Constituents from Raw Materials Used in the Study The flavones, PACs (phenolcarboxylic acids) and total phenolic content were determined using spectrophotometric methods, as follows: Preparation of the extractive solutions: 1.0000 g of vegetable products are refluxed with 50 mL of 50/70% ethanol (in order to establish the optimal concentration of solubilized active principles in the extraction solvent) for 30 min. The extractive solution is obtained by filtration in a 50 mL flask. It’s brought to the mark with the same solvent. Those solutions are used for all types of determinations. 2.3.1. Dosing the Flavonoids Content (FL) The flavonosides and their aglycones form, in the presence of aluminum chloride (neutral medium) and sodium acetate, products of reaction (chelates) with a yellow coloration and more intense fluorescence than the initial compounds. Reagents and solvents: aqueous solutions of sodium acetate 100 g/L and aluminum chloride 25 g/L, rutoside (purchased from Sigma-Aldrich, Schnelldorf, Germany). Carrying out the determination: volumes between 0.3–1.2 mL of extractive solution are placed in a 10 mL volumetric flask, treated with 2 mL of sodium acetate 100 g/L solution and 1 mL of aluminum chloride 25 g/L solution, then it is completed up to the mark with extractive ethanol. In parallel, a control sample is prepared, containing 1 mL of extractive solution and ethanol up to 10 mL. The absorbance of the sample is measured in comparation with the control one at the wavelength λ = 427 nm (maximum determined for metal ion chelation), on a 2005 Jasco V-530 spectrophotometer [ 23 ]. To determine the flavones’ content, the previously constructed standard curve is used, using the following calculation formula: c % = E p E e t × C e t C p × 100 where: c % = the flavonoid concentration of the sample (μg rutozid/100 g dried vegetable product); E p = sample absorbance; E et = the absorbance of the rutoside solution of a known concentration from the standard curve; C et = the concentration of the rutoside solution corresponding to the measured absorbance (μg/mL); C p = mass of dry vegetable product corresponding to 1 mL of sample solution used for dosing (g/mL). A standard rutoside curve was previously established ( Scheme 1 ). The technique used is that described for plant products, replacing the extractive solution with that of rutoside. 2.3.2. Dosing PAC (Phenolic Acids) Phenolic acids react with nitric acid (released after the reaction between sodium nitrite and hydrochloric acid) to form nitroso derivatives, which spontaneously isomerize to isonitroso derivatives. Isonitroso derivatives, acidic compounds, are solubilized in an alkaline medium with the formation of red oxime. Reagents and solvents: aqueous solutions of Arnow’s reagent (sodium nitrite 100 g/L), hydrochloric acid 0.5 N, sodium hydroxide 85 g/L, chlorogenic acid (purchased from Sigma-Aldrich, Schnelldorf, Germany). Carrying out the determination: volumes of extractive solution between 0.2–1 mL are successively treated with 2 mL hydrochloric acid 0.5 M, 2 mL Arnow’s reagent and 2 mL sodium hydroxide 85 g/L, then it is brought up to the mark with distilled water. In parallel, a control is prepared, with a content similar to the sample, except for the Arnow’s reagent. The absorbance of the sample is measured in comparation with a control at the wavelength λ = 525 nm (maximum determined for chlorogenic acid oxime), on a 2005 Jasco V-530 spectrophotometer [ 23 ]. The determination of the total phenol-carboxylic derivatives was determined by interpolation on the chlorogenic acid standard curve. The same calculation formula as the previous type of active principles was also used here. A standard chlorogenic acid curve was previously determined ( Scheme 2 ). 2.3.3. Dosing the Total Phenolic Content (TPC) The phenolic groups reduce the molybdenum derivatives Mo 6+ (colored in yellow) to molybdenum Mo 4+ and Mo 5+ (colored in blue), whose intensity is directly proportional to the concentration. Reagents and solvents: Phosphotungstic acid (PTA), Folin–Ciocâlteu reagent, sodium carbonate 150 g/L, sodium carbonate 200 g/L, and tannic acid (purchased from Sigma-Aldrich, Schnelldorf, Germany). Performing the determination: volumes between 0.1–0.9 mL are diluted to 1 mL with water, treated with 1 mL of Folin–Ciocâlteu reagent and 8 mL of sodium carbonate 200 g/L. The control sample is obtained by replacing the extractive solution with water, adding 1 mL of Folin–Ciocâlteu reagent and 8 mL of sodium carbonate solution 200 g/L. The samples are incubated in the dark for 40 min. Subsequently, the absorbances at λ = 725 nm (the maximum determined for tannic acid), compared to the control samples, on a 2005 Jasco V-530 spectrophotometer [ 23 ]. To determine the total phenolic content, the same calculation formula was used, as above. A standard of total phenolic curve was previously determined ( Scheme 3 ). 2.4. Dosing Volatile Oil Principle of the method: the property of volatile oil to be distillable with water vapor. Steam distillation can be performed in a regular distillation apparatus (open circuit) or in a NeoClevenger type apparatus (closed circuit). Working technique: 50 g of vegetable product and 500–1000 mL of solvent (usually water) are placed in an extraction flask adapted to the apparatus. The mixture thus obtained is subjected to heating directly in the flame, on a sieve. By reaching the boiling point of the extraction solvent, the volatile oil is distilled and captured in the graduated tube of the apparatus. 2.5. Statistical Analysis Statistical analysis was implemented using the open-source software R (R version 4.1.1.) [ 24 ]. Our study contains data sets with small and unequal samples and this fact contradicts with the classical manner so, we use a similar parametric framework but with a bootstrap approach, without concerns about basic violation about normality, homoscedasticity and sphericity [ 25 ]. For simultaneous evaluation, the effect of two factors: Compound (with three levels: FL, PCAss and TPC) and the Sample (with two levels: common crop and control crop) on a response variable named Concentration, we used a two-way robust ANOVA test for every Plant product [ 26 ]. Statistical significance is set to 0.05 (5%) and for post hoc analysis a Bonferroni-adjusted alpha level. 3. Results Plant Material The best results were obtained in the case of a common crop cultivation of two medicinal plants with a wide therapeutic use— Mentha × piperita L. and Melissa officinalis L. ( Figure 2 ). At the time of introduction into the culture, the seedlings did not exceed 10 cm, but over the years they have developed and reached heights of 57 cm for mint and 89 cm for lemon balm [ 23 ]. Following the comparison of the phytosociological crop with the control one, the differences are obvious in terms of vertical and horizontal development dynamics for both species. Drawing a parallel between the crops in May and June 2018 with those in 2019, an abundance in the evolution of plants grown in the same crop, and especially for lemon balm, was observed [ 24 ]. We correlated this fact with an increase of precipitation (215 mm in 2019 compared to 209 mm in 2018), a maintaining of relative humidity at the same average values → 72, and a decrease of 1.3 degrees of maximum temperature recorded ( Table 1 ). For the 2020–2021, the phytosociological crop has taken a different direction, with an increase in density for mint ( Figure 2 f and Figure 3 h). From the point of view of an organoleptic analysis, it was possible to observe during 2018–2020 a significant development and growth of lemon balm in the phytosociological batch ( Figure 2 h), but in 2021 there is a visible difference, with the extension of mint to the detriment of lemon balm ( Figure 3 h). That result it can be associate with a temperature difference (maximum wave → 36.8 °C in 2021 compared to 33.1 °C in 2019), or of lower climatic conditions in precipitation compared to previous years (135 mm in 2021 compared to 215 in 2019) ( Table 1 and Table 2 ). The horizontal evolution of the phytosociological crop mint-lemon balm for 2018–2021 led not only to an increase in the mass of plant product, but also to an overlap between species ( Figure 3 g,h). For a better evaluation, Figure 4 shows the evolutionary stages of Mentha xpiperita L. and Melissa officinalis L. growth in common crops compared with control ones during 2018–2021. The research was extended in parallel to other species of medicinal plants, being cultivated together as follows: Thymus vulgaris L.– Calendula officinalis L. ( Figure 5 ), Rosmarinus officinalis L.– Matricaria chamomilla L. ( Figure 6 a–c), Hypericum perforatum L.– Chelidonium majus L. ( Figure 7 a–c). All phytosociological lots ( Figure 8 were compared with control lots, being grown under the same conditions. Since the association between rosemary and chamomile did not lead to the expected results, we decided to introduce into the culture the association between rosemary and thyme during 2020–2021 ( Figure 6 d–f). Considering that the St. John’s Wort has a different period of development compared to the hollyhock ( Figure 7 a–c), we decided to set up a new batch for further research. Therefore, in 2020, we cultivated St. John’s Wort with another medicinal plant in a common crop– Melissa officinalis L. ( Figure 7 d–f). Figure 8 represents a panoramic characterization of the evolution of the crops in 2019–2021, and Figure 9 shows graphically the evolution of the rest of medicinal plants for the entire study period. In order to observe the morphological changes and certain anatomical features, we also used a macro and microscopic examination using a stereomicroscope (ZEISS Stemi 508 Greenough Stereo Microscope with 8:1 Zoom, details up to 50× magnification) in which we analyzed different types of plant organs (flowers, stems, and leaves). After analyzing the freshly harvested plant material, differences were found between the plant product from the phytosociological crop and from the control one. In the samples of mint and lemon balm (flowers and leaves) there were numerous octocellular glandular hairs that accumulate volatile oil compared to the control crop, where they were less widespread; for the lemon balm in the phytosociological crop, there were crowded and more developed tector hairs ( Figure 10 ). In the thyme leaves and flowers grown in a common crop with marigolds, a higher density of glandular bristles can be observed, which would imply a higher secretion of volatile oil. In the marigold flowers from the common sample, an abundance of cells containing stratified pigment are found, possibly tetraterpenic derivatives of a carotenoid type ( Figure 11 ) [ 28 ]. The rosemary-thyme group had the following changes - the rosemary leaf had a higher number of glandular hairs with volatile oil than the control crop, and the thyme leaf and flower exhibited similar changes ( Figure 12 ). The leaves and petals of St. John’s Wort flowers in the studied crop show a higher number of secretory bags with hypericin (expressed by an increased number of black dots located in the upper peripheral area), and lemon balm leaves have a visibly higher number of octocellular glandular hairs ( Figure 13 ) compared to the control crop. Another important difference appeared in the yarrow-chamomile phytosociological group, with a more obvious horizontal development of the yarrow leaves compared to the control group ( Figure 14 ). From the point of view of the quantity of vegetable product supplied by each lot, there is a significant difference in all the samples analyzed compared to the control ones. ( Figure 15 — Table 3 and Figure 16 — Table 4 ). We want to mention that for mint, lemon balm and rosemary—the leaves were weighed; for thyme, St. John’s Wort, chamomile and yarrow—the upper third of the aerial part and for marigolds—the flowers were weighed. According to the data analysis obtained from Table 3 and Table 4 and following the graphical representation in Figure 16 and Figure 17 , there has been a development in providing the amount of vegetable raw material. It is much more obvious in phytosociological crops other than the control ones. The results of the quantitative chemical determinations are given in Table 5 (volumetric dosing of volatile oil) and Table 6 (dosing of flavones, PCAs and total phenolic content through spectrophotometric methods). These data are represented graphically in Figure 17 (representation of total flavones content) and Figure 18 and Figure 19 constitute the total PCAs content respectively TPC. 4. Discussion In this present research we have been observing this relationship and how a group of medicinal plants can be influenced, by being grown together. This activity took place without intervenion in any way for the common crop such as: enriching the soil with a type of fertilizer, applying different amounts of water than the control one, etc. Morpho-anatomical differences occurred in the distribution of frequency, density, abundance and relative frequency in the common group compared to the control group. Compared to the control crops, it was observed that each combination of medicinal plants leads to the biosynthesis of a larger quantity of secondary metabolites (volatile oil, flavones, PCAs and total polyphenols— Table 5 and Figure 17 , Figure 18 and Figure 19 ). This was also remarked ino the study published on the association between mint and lemon balm, the culture period followed being 2018–2019 [ 23 ]. Based on the statistical analysis, a direct correlation was found in certain types of cultures in terms of the biosynthesis of secondary metabolites. Following the same batches of mint and lemon balm during 2020–2021, showed a net increase in the production of plant raw materials, a fact possibly due to weather conditions, but also much better accommodation in the culture of the two medicinal species. Although they reacted well in different associations, there were also situations of medicinal plants that did not adapt ( Rosmarinus officinalis L. and Matricaria chamomilla L.), perhaps due to the different period of development ( Hypericum perforatum L. and Chelidonium majus L.). These are the observations of our study, unfortunately without correlating with the literature data, as the published data generally refer to the associations of plants that grow in spontaneous flora and forest areas [ 29 , 30 ]. A higher dominance of octocellular glandular bristles in plant products from Lamiaceae family species is correlated according to our research with a slightly higher amount of volatile oil, the quantities being dependent on the nature of the plant’s raw material. The presence of cells with dominant pigments in the epidermal cells derived from petals in marigold flowers is closely correlated with a higher content of flavonic derivatives, yellow pigments. According to the results obtained from the quantitative chemical determinations, it is found that ethanol 50% extracts a larger amount of active ingredients, which can be a starting point in the more detailed phytochemical analyses performed on these plant species. 5. Conclusions Following the study, we can say that the combinations of medicinal plants, either belonging to the same family or different families, are beneficial, as a consequence of their horizontal and vertical development compared to control crops, the amount of plant product provided being much higher, including the anatomical formations that accumulate volatile oil being dominant in these associations. We mention the fact that at the moment we are studying other common crops of mint-lemon balm where we aim to measure the quantity and quality of microelements following the enrichment of the soil with a chemical or biological fertilizer. We will compare them with a control soil. Author Contributions Conceptualization: E.A.L., C.E.G. and M.G. Data curation: E.A.L. and C.E.G.; Formal analysis: E.A.L. and C.E.G.; Investigation: E.A.L., C.E.G. and M.G.; Methodology: E.A.L., C.E.G. and M.G.; Supervision: C.E.G. and M.G.; Validation: C.E.G.; Visualization: E.A.L. and C.E.G.; Writing—original draft preparation: E.A.L. and C.E.G.; Writing—review and editing: E.A.L., C.E.G. and M.G.; Project administration: E.A.L. and C.E.G. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Data Availability Statement MDPI Research Data Policies at https://www.mdpi.com/ethics (5 December 2021). Acknowledgments This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. All individuals included in this section have consented to the acknowledgement. Conflicts of Interest The authors declare that there are no conflict of interest related to this article. References Bacieczko, W.; Kaszycka, E.; Borcz, A. 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Evolutionary stages of Mentha piperita L. and Melissa officinalis L. growth in common crops compared with control ones during 2020–2021 ( a – p ); Legend: h—height; MM—peppermint control crop; MF—peppermint phytosociological (common) crop; MLM—lemon balm control crop; and MLF—lemon balm phytosociological (common) crop. ( a ) MM; h = 32 cm; ( b ) MF; h = 44 cm; ( c ) MLM; h = 34 cm; ( d ) MLF; h = 51 cm; ( e ) MM; h = 48 cm; ( f ) MF; h = 52 cm; ( g ) MLM; h = 55 cm; ( h ) MLF; h = 74 cm; ( i ) MM; h = 57 cm; ( j ) ML; h = 43 cm; ( k ) MLM; h = 50 cm; ( l ) MLF; h= 47 cm; ( m ) MM; h = 80 cm; ( n ) ML; h = 57 cm; ( o ) MLM; h = 70 cm; ( p ) MLF; h = 59 cm. Figure 2. Evolutionary stages of Mentha piperita L. and Melissa officinalis L. growth in common crops compared with control ones during 2020–2021 ( a – p ); Legend: h—height; MM—peppermint control crop; MF—peppermint phytosociological (common) crop; MLM—lemon balm control crop; and MLF—lemon balm phytosociological (common) crop. ( a ) MM; h = 32 cm; ( b ) MF; h = 44 cm; ( c ) MLM; h = 34 cm; ( d ) MLF; h = 51 cm; ( e ) MM; h = 48 cm; ( f ) MF; h = 52 cm; ( g ) MLM; h = 55 cm; ( h ) MLF; h = 74 cm; ( i ) MM; h = 57 cm; ( j ) ML; h = 43 cm; ( k ) MLM; h = 50 cm; ( l ) MLF; h= 47 cm; ( m ) MM; h = 80 cm; ( n ) ML; h = 57 cm; ( o ) MLM; h = 70 cm; ( p ) MLF; h = 59 cm. Figure 3. The horizontal evolution of peppermint and lemon balm phytosociological crops during 2018–2021 ( a – h ). ( a ) Phytosociological crop, May 2018; ( b ) Phytosociological crop, June 2018; ( c ) Phytosociological crop, May 2019; ( d ) Phytosociological crop, June 2019; ( e ) Phytosociological crop, May 2020; ( f ) Phytosociological crop, June 2020; ( g ) Phytosociological crop, May 2021; ( h ) Phytosociological crop, June 2021. Figure 3. The horizontal evolution of peppermint and lemon balm phytosociological crops during 2018–2021 ( a – h ). ( a ) Phytosociological crop, May 2018; ( b ) Phytosociological crop, June 2018; ( c ) Phytosociological crop, May 2019; ( d ) Phytosociological crop, June 2019; ( e ) Phytosociological crop, May 2020; ( f ) Phytosociological crop, June 2020; ( g ) Phytosociological crop, May 2021; ( h ) Phytosociological crop, June 2021. Figure 4. Evolutionary stages of Mentha × piperita L. and Melissa officinalis L. growth in common crops [ 23 ] compared with control ones during 2018–2021; Legend: MM—peppermint control crop; MF—peppermint phytosociological (common) crop; MLM—lemon balm control crop; and MLF—lemon balm phytosociological (common) crop; evolution of heights—y axis. Figure 4. Evolutionary stages of Mentha × piperita L. and Melissa officinalis L. growth in common crops [ 23 ] compared with control ones during 2018–2021; Legend: MM—peppermint control crop; MF—peppermint phytosociological (common) crop; MLM—lemon balm control crop; and MLF—lemon balm phytosociological (common) crop; evolution of heights—y axis. Figure 5. The horizontal evolution of Thymus vulgaris L. and Calendula officinalis L. phytosociological crops during 2018–2021 ( a – f ), where h—height. ( a ) Phytosociological crop thyme (h-23 cm) and marigolds (h-29 cm), May 2018; ( b ) Phytosocio-logical crop thyme (h-29 cm) and marigolds (h-37 cm), June 2018; ( c ) Phytosociological crop thyme (h-16 cm) and marigolds (h-33 cm), May 2019; ( d ) Control group marigolds (h-22 cm) May 2020; ( e ) Phytosociological crop thyme (h-30 cm) and marigolds (h-41 cm), June 2020; ( f ) Phytosociolog-ical crop thyme (h-14 cm) and marigolds (h-16 cm), May 2021. Figure 5. The horizontal evolution of Thymus vulgaris L. and Calendula officinalis L. phytosociological crops during 2018–2021 ( a – f ), where h—height. ( a ) Phytosociological crop thyme (h-23 cm) and marigolds (h-29 cm), May 2018; ( b ) Phytosocio-logical crop thyme (h-29 cm) and marigolds (h-37 cm), June 2018; ( c ) Phytosociological crop thyme (h-16 cm) and marigolds (h-33 cm), May 2019; ( d ) Control group marigolds (h-22 cm) May 2020; ( e ) Phytosociological crop thyme (h-30 cm) and marigolds (h-41 cm), June 2020; ( f ) Phytosociolog-ical crop thyme (h-14 cm) and marigolds (h-16 cm), May 2021. Figure 6. The horizontal evolution of Rosmarinus officinalis L. and Matricaria chamomilla L. phytosociological crops during 2018–2019, changed with Rosmarinus officinalis L. and Thymus vulgaris L. during 2020–2021 ( a – f ), where h—height. ( a ) Phytosociological crop rosemary (h-44 cm) and chamomile (h-24 cm), May 2018; ( b ) Phytoso-ciological crop rosemary (h-49 cm) and chamomile (h-54 cm), May 2019; ( c ) Phytosociological crop rosemary (h-53 cm) and chamomile (h-62 cm), June 2019; ( d ) Phytosociological crop rosemary (h-98 cm) and thyme (h-18 cm), May 2020; ( e ) Phytosociological crop rosemary (h-120 cm) and thyme (h-30 cm), June 2020; ( f ) Phytosociological crop rosemary (h-130 cm) and thyme (h-24 cm), June 2021. Figure 6. The horizontal evolution of Rosmarinus officinalis L. and Matricaria chamomilla L. phytosociological crops during 2018–2019, changed with Rosmarinus officinalis L. and Thymus vulgaris L. during 2020–2021 ( a – f ), where h—height. ( a ) Phytosociological crop rosemary (h-44 cm) and chamomile (h-24 cm), May 2018; ( b ) Phytoso-ciological crop rosemary (h-49 cm) and chamomile (h-54 cm), May 2019; ( c ) Phytosociological crop rosemary (h-53 cm) and chamomile (h-62 cm), June 2019; ( d ) Phytosociological crop rosemary (h-98 cm) and thyme (h-18 cm), May 2020; ( e ) Phytosociological crop rosemary (h-120 cm) and thyme (h-30 cm), June 2020; ( f ) Phytosociological crop rosemary (h-130 cm) and thyme (h-24 cm), June 2021. Figure 7. The horizontal evolution of Hypericum perforatum L. and Chelidonium majus L. phytosociological crops during 2018–2019 ( a – f ), changed with Hypericum perforatum L. and Melissa officinalis L. during 2020–2021, where h—height. ( a ) Phytosociological crop St. John’s Wort (h-60 cm) and celandine (h-25 cm), May 2018; ( b ) Phy-tosociological crop St. John’s Wort (h-73 cm) and celandine (h-52 cm), May 2019; ( c ) Phytosocio-logical crop St. John’s Wort (h-93 cm) and celandine (h-58 cm) June 2019; ( d ) Phytosociological crop St. John’s Wort (h-74 cm) and lemon balm (h-28 cm), May 2020; ( e ) Phytosociological crop St. John’s Wort (h-78 cm) and lemon balm (h-52 cm), June 2020; ( f ) Phytosociological crop St. John’s Wort (h-82 cm) and lemon balm (h-56 cm), June 2021. Figure 7. The horizontal evolution of Hypericum perforatum L. and Chelidonium majus L. phytosociological crops during 2018–2019 ( a – f ), changed with Hypericum perforatum L. and Melissa officinalis L. during 2020–2021, where h—height. ( a ) Phytosociological crop St. John’s Wort (h-60 cm) and celandine (h-25 cm), May 2018; ( b ) Phy-tosociological crop St. John’s Wort (h-73 cm) and celandine (h-52 cm), May 2019; ( c ) Phytosocio-logical crop St. John’s Wort (h-93 cm) and celandine (h-58 cm) June 2019; ( d ) Phytosociological crop St. John’s Wort (h-74 cm) and lemon balm (h-28 cm), May 2020; ( e ) Phytosociological crop St. John’s Wort (h-78 cm) and lemon balm (h-52 cm), June 2020; ( f ) Phytosociological crop St. John’s Wort (h-82 cm) and lemon balm (h-56 cm), June 2021. Figure 8. The evolution and dynamics of the whole culture during our study. Figure 8. The evolution and dynamics of the whole culture during our study. Figure 9. Evolutionary stages of the phytosociological culture; Legend: Thyme and Marigold (TM)— Thymus vulgaris L.– Calendula officinalis L. crop, Rosemary and Chamomile (RC)— Rosmarinus officinalis L.– Matricaria chamomilla L. crop, Rosemary and Thyme (RT)— Rosmarinus officinalis L– Thymus vulgaris L. crop, St. John’s and Celadine (SC)— Hypericum perforatum L.– Chelidonium majus L. crop, St. John’s and Lemon Balm (SL)— Hypericum perforatum L.– Melissa officinalis L. crop; evolution of heights—y axis. Figure 10. Mint—Lemon balm. Figure 10. Mint—Lemon balm. Figure 11. Thyme—Marigold. Figure 11. Thyme—Marigold. Figure 12. Rosemary—Thyme. Figure 12. Rosemary—Thyme. Figure 13. St. John’s Wort—Lemon Balm. Figure 13. St. John’s Wort—Lemon Balm. Figure 14. Yarrow. Figure 14. Yarrow. Figure 15. Graphic representation of vegetable products’ mass in 2020. Figure 16. Graphic representation of vegetable products mass in 2021. Figure 16. Graphic representation of vegetable products mass in 2021. Figure 17. Graphic representation of total flavones content. Figure 17. Graphic representation of total flavones content. Figure 18. Graphic representation of total PACs (phenolcarboxylic acids) content. Figure 18. Graphic representation of total PACs (phenolcarboxylic acids) content. Figure 19. Graphic representation of total phenolic content. As we highlight in the figures for the each Plant product ( Figure 20 a – j ) there is a statistically significant interaction ( p value < 0.05) between the effects of Compound and Sample on value of concentration except four cases: MT, TM, ML and YC. Simple main effects analysis showed that the common crop is statistically different from control crop ( p value < 0.05), except YC and between FL, PCA’s and TPC there are statistical differences, except TR, TM, ML and YC where PCA’s and TPC behave similarly statistically. Figure 19. Graphic representation of total phenolic content. As we highlight in the figures for the each Plant product ( Figure 20 a – j ) there is a statistically significant interaction ( p value < 0.05) between the effects of Compound and Sample on value of concentration except four cases: MT, TM, ML and YC. Simple main effects analysis showed that the common crop is statistically different from control crop ( p value < 0.05), except YC and between FL, PCA’s and TPC there are statistical differences, except TR, TM, ML and YC where PCA’s and TPC behave similarly statistically. Figure 20. Statistical analysis a two-way interaction boxplot for medicinal plant. ( a ) A two-way interaction boxplot for TR; ( b ) A two-way interaction boxplot for RT; ( c ) A two-way interaction boxplot for SL; ( d ) A two-way interaction boxplot for LS; ( e ) A two-way in-teraction boxplot for MT; ( f ) A two-way interaction boxplot for TM; ( g ) A two-way interaction boxplot for ML; ( h ) A two-way interaction boxplot for LM; ( i ) A two-way interaction boxplot for CY; ( j ) A two-way interaction boxplot for YC. Figure 20. Statistical analysis a two-way interaction boxplot for medicinal plant. ( a ) A two-way interaction boxplot for TR; ( b ) A two-way interaction boxplot for RT; ( c ) A two-way interaction boxplot for SL; ( d ) A two-way interaction boxplot for LS; ( e ) A two-way in-teraction boxplot for MT; ( f ) A two-way interaction boxplot for TM; ( g ) A two-way interaction boxplot for ML; ( h ) A two-way interaction boxplot for LM; ( i ) A two-way interaction boxplot for CY; ( j ) A two-way interaction boxplot for YC. Table 1. Climatic conditions in Turnu Magurele town (Romania) during 2018–2019 according to ANM (National Weather Service) [ 27 ]. Table 1. Climatic conditions in Turnu Magurele town (Romania) during 2018–2019 according to ANM (National Weather Service) [ 27 ]. Turnu Magurele Period Medium Value Minim Value (Date) Maxim Value (Date) Number of Observations T air (°C) at altitudes of 2 m above the ground 01.05–30.06.2018 +20.9 +8.8 (13.05.2018) +34.4 (13.06.2018) 1452 01.05–30.06.2019 +20.1 +5.6 (09.05.2019) +33.1 (23.06.2019) 1461 P0, atmospheric pressure at the station level (mmHg) 01.05–30.06.2018 756.9 747.5 (30.06.2018) 764.0 (28.05.2018) 1452 01.05–30.06.2019 757.3 747.8 (05.05.2019) 765.5 (26.06.2019) 1461 U, relative humidity (%), 2 m above the ground 01.05–30.06.2018 72 24 (29.05.2018) 1452 01.05–30.06.2019 72 25 (03.05.2019) 1461 The amount of precipitation Maxim Value (date) The proportion of days with precipitation Number of observations RRR, the amount of precipitation (milimeters) 01.05–30.06.2018 209 50.0 in 12 h (28.06.2018) 31 121 01.05–30.06.2019 215 42.0 in 12 h (25.06.2019) 24 122 Table 2. Climatic conditions in Turnu Magurele town (Romania) during 2020–2021 according to ANM (National Weather Service) [ 27 ]. Table 2. Climatic conditions in Turnu Magurele town (Romania) during 2020–2021 according to ANM (National Weather Service) [ 27 ]. Turnu Magurele Period Medium Value Minim Value (Date) Maxim Value (Date) Number of Observations T air (°C) at altitudes of 2 m above the ground 01.05–30.06.2020 +19.2 +6.5 (09.05.2020) +33.5 (29.06.2020) 1464 01.05–30.06.2021 +19.5 +4.5 (09.05.2021) +36.8 (25.06.2021) 1464 P0, atmospheric pressure at the station level (mmHg) 01.05–30.06.2020 756.7 749.2 (02.05.2020) 765.6 (23.05.2020) 1464 01.05–30.06.2021 757.8 750.6 (13.05.2021) 767.5 (09.05.2021) 1464 U, relative humidity (%), 2 m above the ground 01.05–30.06.2020 68 22 (28.06.2020) 1464 01.05–30.06.2021 69 23 (12.05.2021) 1464 The amount of precipitation Maxim Value (date) The proportion of days with precipitation Number of observations RRR, the amount of precipitation (milimeters) 01.05–30.06.2020 163 35.0 in 12 h (16.06.2020) 23 122 01.05–30.06.2021 135 22.0 in 12 h (25.06.2019) 25 122 Table 3. Representation of vegetable products’ mass in 2020. Table 3. Representation of vegetable products’ mass in 2020. Plant Products Control Crop Common Crop g Harvested g Dry g Harvested g Dry thyme (thyme-rosemary) 540 283 730 302 rosemary (thyme-rosemary) 3524 1200 4920 2107 St. John’s Wort (St. John’s Wort—lemon balm) 1423 697 1934 895 lemon balm (St. John’s Wort—lemon balm) 480 193 228 102 marigold (marigold-thyme) 208 96 300 123 thyme (marigold-thyme) 540 283 789 325 mint (mint-lemon balm) 1200 543 2492 982 lemon balm (mint-lemon balm) 480 193 2430 1010 chamomile (yarrow-chamomile) 356 152 543 267 yarrow (yarrow-chamomile) 324 146 403 185 Table 4. Representation of vegetable products’ mass in 2021. Table 4. Representation of vegetable products’ mass in 2021. Plant Products Control Crop Common Crop g Harvested g Dry g Harvested g Dry thyme (thyme-rosemary) 970 403 1230 450 rosemary (thyme-rosemary) 4409 1878 6830 3104 St. John’s Wort (St. John’s Wort—lemon balm) 1800 823 2700 1094 lemon balm (St. John’s Wort—lemon balm) 595 223 480 150 marigold (marigold-thyme) 700 350 700 350 thyme (marigold-thyme) 970 403 1300 518 mint (mint-lemon balm) 4360 1800 4075 1650 lemon balm(mint-lemon balm) 595 223 810 314 chamomile (yarrow-chamomile) 469 210 470 125 yarrow (yarrow-chamomile) 570 200 740 305 Table 5. Determining the amount of essential oil in certain vegetable products. Table 5. Determining the amount of essential oil in certain vegetable products. Table 6. Results for spectrophotometric and volumetric assay for every medicinal plant in the culture. Table 6. Results for spectrophotometric and volumetric assay for every medicinal plant in the culture. g FL Expressed in Rutin/100 g Dried Leaves g PCAs Expressed in Chlorogenic Acid/100 g Dried Leaves g TPC Expressed in Tannic Acid/100 g Dried Leaves Plant Product Solvent Control Crop Common Crop Control Crop Common Crop Control Crop Common Crop Alcohol 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 TR 70% - 1.9317 ± 0.0947 - 2.4413 ± 0.1858 - - - - - - - - 50% - - - - - 6.9709 ± 1.4921 - 9.9461 ± 0.8385 - 6.0393 ± 0.3204 - 11.1911 ± 0.7959 RT 70% 1.2555 ± 0.3082 1.3469 ± 0.1941 1.5908 ± 0.1292 1.7616 ± 0.1322 9.7633 ± 0.3391 10.0288 ± 0.4307 11.659 ± 1.1725 13.0085 ± 0.5305 10.0337 ± 0.2470 10.2605 ± 0.4612 13.6982 ± 3.4303 14.3533 ± 3.4511 50% 1.6612 ± 0.2336 - 1.7626±0.2195 - 10.312 ± 1.4714 - 11.2637 ± 1.4027 - 9.2616 ± 0.3351 - 11.0854 ± 0.2787 - SL 70% - - - - - - - - - - - - 50% - 4.3646 ± 1.4447 - 6.1703 ± 1.1658 - 16.6146 ± 1.0430 - 20.9229 ± 0.9239 - 6.7989 ± 0.3940 - 8.1598 ± 0.4262 LS 70% - 1.6432 ± 0.2505 - 2.1422 ± 0.5379 - 11.8405 ± 0.7671 - 22.0896 ± 1.5231 - 3.614 ± 0.421 - 5.9761 ± 0.0938 50% - - - - - - - - - - - - MT 70% 0.787 ± 0.1351 0.6944 ± 0.0805 1.1311 ± 0.0578 1.1504 ± 0.0643 - - - - 4.0237 ± 0.8222 - 4.2247 ± 1.6928 - 50% 0.6376 ± 0.0505 - 1.0759 ± 0.0951 - 1.3438 ± 0.0999 1.4104 ± 0.1216 1.5514 ± 0.1935 2.0048 ± 0.2633 3.1223 ± 0.2800 3.3329 ± 0.4030 3.4311 ± 0.7578 4.1516 ± 0.5974 TM 70% 2.0462 ± 0.5865 1.9317 ± 0.0947 2.6249 ± 1.1390 2.3134 ± 0.4572 8.3479 ± 1.3352 - 8.9926 ± 1.0686 - 10.556 ± 1.3394 - 11.639 ± 2.2604 - 50% 2.0646 ± 0.2753 - 2.5947 ± 0.0961 - 6.2302 ± 0.9905 6.9709 ± 1.4921 7.5046 ± 0.2743 8.2233 ± 0.5946 5.8147 ± 1.0630 6,0393 ± 0.3204 9.2512 ± 1.3221 10.3147 ± 1.2546 ML 70% - - - - - - - - - 9.1505 ± 1.9447 - 12.041 ± 1.9260 50% - 1.024 ± 0.2407 - 2.2621 ± 0.1475 - 9.5829 ± 1.0670 - 12.0579 ± 0.7928 - - - - LM 70% - 1.6432 ± 0.2505 - 2.2951 ± 0.7055 - 11.8405 ± 0.7671 - 19.6639 ± 2.6681 - 3.614 ± 0.421 - 6.4694 ± 0.5147 50% - - - - - - - - - - - - CY 70% 1.7606 ± 0.1229 - 1.8712 ± 0.1004 - 1.7185 ± 0.2359 - 2.1868 ± 0.2834 - 4.7869 ± 0.6933 4.8703 ± 1.1159 50% 1.8272 ± 0.5233 1.8057 ± 0.5497 2.0404 ± 0.6936 2.4074 ± 1.3468 1.629 ± 0.2360 1.682 ± 0.2470 1.9253 ± 0.1624 2.6278 ± 0.3760 2.8405 ± 0.2988 2.8963 ± 0.3025 2.9047 ± 0.2621 3.5099 ± 0.2954 YC 70% - - - - - - - - - - - - 50% - 1.5354 ± 0.1772 - 2.5909 ± 0.4796 - 12.4033 ± 4.5895 - 13.8511 ± 4.3555 - 11.1061 ± 0.8620 - 13.7817 ± 2.9323 Results are mean ± SD ( n = 3); FL—flavones; PCAs—phenolcarboxylic acids; TPC—total phenolic content; Legend: TR—thyme (thyme-rosemary); RT—rosemary (thyme-rosemary); SL-St. John’s Wort (St. John’s Wort—lemon balm); LS—lemon balm (St. John’s Wort—lemon balm); MT—marigold (marigold-thyme); TM—thyme (marigold-thyme); ML—mint (mint-lemon balm); LM—lemon balm(mint-lemon balm); CY—chamomile (yarrow-chamomile); YC—yarrow (yarrow-chamomile). Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Article Metrics MDPI and ACS Style Luță, E.A.; Ghica, M.; Gîrd, C.E. The Initiation of a Phytosociological Study on Certain Types of Medicinal Plants. Agriculture 2022, 12, 283. https://doi.org/10.3390/agriculture12020283 AMA Style Luță EA, Ghica M, Gîrd CE. The Initiation of a Phytosociological Study on Certain Types of Medicinal Plants. Agriculture. 2022; 12(2):283. https://doi.org/10.3390/agriculture12020283 Chicago/Turabian Style Luță, Emanuela Alice, Manuela Ghica, and Cerasela Elena Gîrd. 2022. "The Initiation of a Phytosociological Study on Certain Types of Medicinal Plants" Agriculture12, no. 2: 283. https://doi.org/10.3390/agriculture12020283 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here .	https://www.mdpi.com/2077-0472/12/2/283/htm
U.S. Census Bureau QuickFacts: Vermont; United States; Keya Paha County, Nebraska; Colfax County, Nebraska Frequently requested statistics for: Vermont; United States; Keya Paha County, Nebraska; Colfax County, Nebraska QuickFacts Vermont; United States; Keya Paha County, Nebraska; Colfax County, Nebraska QuickFacts provides statistics for all states and counties, and for cities and towns with a population of 5,000 or more. Table <table><tbody><tr><th> All Topics Population Age and Sex Race and Hispanic Origin Population Characteristics Housing Families & Living Arrangements Computer and Internet Use Education Health Economy Transportation Income & Poverty Businesses Geography</th><th> Vermont</th><th> United States</th><th> Keya Paha County, Nebraska</th><th> Colfax County, Nebraska</th></tr><tr><td> Population Estimates, July 1, 2022, (V2022)</td><td>  647,064</td><td>  333,287,557</td><td>  787</td><td>  10,444</td></tr></tbody></table> People Population Population Estimates, July 1, 2022, (V2022)  647,064  333,287,557  787  10,444 Population estimates base, April 1, 2020, (V2022)  643,085  331,449,520  768  10,581 Population, percent change - April 1, 2020 (estimates base) to July 1, 2022, (V2022)  0.6%  0.6%  2.5%  -1.3% Population, Census, April 1, 2020643,077331,449,28176910,582 Population, Census, April 1, 2010625,741308,745,53882410,515 Age and Sex Persons under 5 years, percent  4.3%  5.6%  3.9%  8.5% Persons under 18 years, percent  17.7%  21.7%  18.7%  29.9% Persons 65 years and over, percent  21.6%  17.3%  32.8%  14.3% Female persons, percent  50.2%  50.4%  47.9%  46.2% Race and Hispanic Origin White alone, percent  93.8%  75.5%  98.0%  87.2% Black or African American alone, percent (a)  1.5%  13.6%  0.0%  5.4% American Indian and Alaska Native alone, percent (a)  0.4%  1.3%  0.9%  3.8% Asian alone, percent (a)  2.1%  6.3%  0.4%  1.3% Native Hawaiian and Other Pacific Islander alone, percent (a)   Z  0.3%  0.0%  0.7% Two or More Races, percent  2.1%  3.0%  0.8%  1.6% Hispanic or Latino, percent (b)  2.3%  19.1%  0.8%  47.0% White alone, not Hispanic or Latino, percent  91.9%  58.9%  97.6%  46.7% Population Characteristics Veterans, 2017-202134,34717,431,29040488 Foreign born persons, percent, 2017-20214.4%13.6%2.3%34.5% Housing Housing units, July 1, 2022, (V2022)339,034143,786,6554954,001 Owner-occupied housing unit rate, 2017-202172.1%64.6%80.0%76.7% Median value of owner-occupied housing units, 2017-2021$240,600$244,900$85,500$89,700 Median selected monthly owner costs -with a mortgage, 2017-2021$1,701$1,697$1,477$1,158 Median selected monthly owner costs -without a mortgage, 2017-2021$724$538$402$448 Median gross rent, 2017-2021$1,070$1,163$767$749 Building permits, 20222,3021,665,088018 Families & Living Arrangements Households, 2017-2021262,514124,010,9923303,582 Persons per household, 2017-20212.352.602.872.94 Living in same house 1 year ago, percent of persons age 1 year+, 2017-202187.1%86.6%96.3%94.0% Language other than English spoken at home, percent of persons age 5 years+, 2017-20215.5%21.7%4.4%47.9% Computer and Internet Use Households with a computer, percent, 2017-202192.3%93.1%85.5%93.3% Households with a broadband Internet subscription, percent, 2017-202184.6%87.0%82.1%85.5% Education High school graduate or higher, percent of persons age 25 years+, 2017-202193.9%88.9%95.3%73.4% Bachelor's degree or higher, percent of persons age 25 years+, 2017-202140.9%33.7%27.8%15.1% Health With a disability, under age 65 years, percent, 2017-202110.4%8.7%4.5%6.5% Persons without health insurance, under age 65 years, percent  4.5%  9.8%  8.8%  19.0% Economy In civilian labor force, total, percent of population age 16 years+, 2017-202164.9%63.1%63.9%69.8% In civilian labor force, female, percent of population age 16 years+, 2017-202162.8%58.7%53.9%63.6% Total accommodation and food services sales, 2017 ($1,000) (c)2,019,843938,237,077 NA D Total health care and social assistance receipts/revenue, 2017 ($1,000) (c)5,451,6262,527,903,275 NA28,197 Total transportation and warehousing receipts/revenue, 2017 ($1,000) (c)772,885895,225,411 NA D Total retail sales, 2017 ($1,000) (c)10,811,2814,949,601,4813,36955,867 Total retail sales per capita, 2017 (c)$17,294$15,224$4,265$5,221 Transportation Mean travel time to work (minutes), workers age 16 years+, 2017-202123.326.816.318.1 Income & Poverty Median household income (in 2021 dollars), 2017-2021$67,674$69,021$54,583$70,381 Per capita income in past 12 months (in 2021 dollars), 2017-2021$37,903$37,638$32,282$26,972 Persons in poverty, percent  10.3%  11.6%  19.3%  8.3% <table> Businesses<tbody><tr><th> Businesses</th><th></th><th></th><th></th><th></th></tr><tr><td> Total employer establishments, 2021</td><td>20,696</td><td>8,148,606</td><td>16</td><td>250</td></tr><tr><td> Total employment, 2021</td><td>239,758</td><td>128,346,299</td><td>42</td><td>3,980</td></tr><tr><td> Total annual payroll, 2021 ($1,000)</td><td>12,710,835</td><td>8,278,573,947</td><td>1,065</td><td>198,506</td></tr><tr><td> Total employment, percent change, 2020-2021</td><td>-7.2%</td><td>-4.3%</td><td>0.0%</td><td>0.6%</td></tr><tr><td> Total nonemployer establishments, 2019</td><td>61,921</td><td>27,104,006</td><td>118</td><td>552</td></tr><tr><td> All employer firms, Reference year 2017</td><td>17,206</td><td>5,744,643</td><td> S</td><td>283</td></tr><tr><td> Men-owned employer firms, Reference year 2017</td><td>9,255</td><td>3,480,438</td><td> S</td><td>157</td></tr><tr><td> Women-owned employer firms, Reference year 2017</td><td>2,649</td><td>1,134,549</td><td> S</td><td> S</td></tr><tr><td> Minority-owned employer firms, Reference year 2017</td><td>417</td><td>1,014,958</td><td> S</td><td> S</td></tr><tr><td> Nonminority-owned employer firms, Reference year 2017</td><td>14,640</td><td>4,371,152</td><td> S</td><td>233</td></tr><tr><td> Veteran-owned employer firms, Reference year 2017</td><td>921</td><td>351,237</td><td> S</td><td> S</td></tr><tr><td> Nonveteran-owned employer firms, Reference year 2017</td><td>13,703</td><td>4,968,606</td><td> S</td><td>201</td></tr></tbody></table> <table> Geography<tbody><tr><th> Geography</th><th></th><th></th><th></th><th></th></tr><tr><td> Population per square mile, 2020</td><td>69.8</td><td>93.8</td><td>1.0</td><td>25.7</td></tr><tr><td> Population per square mile, 2010</td><td>67.9</td><td>87.4</td><td>1.1</td><td>25.5</td></tr><tr><td> Land area in square miles, 2020</td><td>9,217.45</td><td>3,533,038.28</td><td>773.08</td><td>411.65</td></tr><tr><td> Land area in square miles, 2010</td><td>9,216.66</td><td>3,531,905.43</td><td>773.07</td><td>411.66</td></tr><tr><td> FIPS Code</td><td>50</td><td>1</td><td>31103</td><td>31037</td></tr></tbody></table> About datasets used in this table Value Notes  Estimates are not comparable to other geographic levels due to methodology differences that may exist between different data sources. Some estimates presented here come from sample data, and thus have sampling errors that may render some apparent differences between geographies statistically indistinguishable. ] Click the Quick Info icon to the left of each row in TABLE view to learn about sampling error. In Vintage 2022, as a result of the formal request from the state, Connecticut transitioned from eight counties to nine planning regions. For more details, please see the Vintage 2022 release notes available here:Release Notes. The vintage year (e.g., V2022) refers to the final year of the series (2020 thru 2022). Different vintage years of estimates are not comparable. Users should exercise caution when comparing 2017-2021 ACS 5-year estimates to other ACS estimates. For more information, please visit the2021 5-year ACS Comparison Guidancepage. Fact Notes (a)Includes persons reporting only one race (c)Economic Census - Puerto Rico data are not comparable to U.S. Economic Census data (b)Hispanics may be of any race, so also are included in applicable race categories Value Flags -Either no or too few sample observations were available to compute an estimate, or a ratio of medians cannot be calculated because one or both of the median estimates falls in the lowest or upper interval of an open ended distribution. FFewer than 25 firms DSuppressed to avoid disclosure of confidential information NData for this geographic area cannot be displayed because the number of sample cases is too small. FNFootnote on this item in place of data XNot applicable SSuppressed; does not meet publication standards NANot available ZValue greater than zero but less than half unit of measure shown	https://www.census.gov/quickfacts/fact/table/VT,US,keyapahacountynebraska,colfaxcountynebraska/PST045222
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947f2189054e0269d0d3ea65684f9816460d28fcd988d473204642457fbe3a64.pdf 5-64. 2009 Chevrolet Silverado Owner Manual M ... A French language copy of this manual can be obtained ... If the vehicle has a manual seat, it can be moved. GM Owner Manuals EDS 2009 Chevrolet Silverado Owner Manual M Keep this manual in the vehicle for quick reference. Canadian Owners Canadian Owners (Propriétaires Canadiens) A French language copy of this manual can be obtained from your dealer/retailer or from: On peut obtenir un exemplaire de ce guide en français auprès du concessionnaire ou à l’adresse suivante: Helm, Incorporated P.O. Box 07130 Owners Manual - dealereprocess.net 5-64. 2009 Chevrolet Silverado Owner Manual M ... A French language copy of this manual can be obtained ... If the vehicle has a manual seat, it can be moved. . If the position you are using has an adjustable headrest or head restraint and you are using a single tether, raise the headrest or head restraint and route the tether under the headrest or head restraint and in between the headrest or head restraint posts. 1-55 2. See Securing a Child Restraint in the Right Front Seat Position (With Airbag Off Switch) on page 1-62 or Securing a Child Restraint in the Right Front Seat Position (With Passenger Sensing System) on page 1-66 or Securing a Child Restraint in the Right Front Seat Position (Heavy Duty Crew Cab Only) on page 1-70 for instructions on installing the child restraint using the safety belts. 3. Push and pull the child restraint in different directions to be sure it is secure. Crew and Extended Cab Models 1. Attach and tighten the lower attachments to the lower anchors. If the child restraint does not have lower attachments or the desired seating position does not have lower anchors, secure the child restraint with the top tether and the safety belts. Refer to your child restraint manufacturer instructions and the instructions in this manual. 1.1. Find the lower anchors for the desired seating position. 1.2. Put the child restraint on the seat. 1.3. Attach and tighten the lower attachments on the child restraint to the lower anchors. 2. If the child restraint manufacturer recommends that the top tether be attached, attach and tighten the top tether to the top tether anchor (loop), if your vehicle has one. Refer to the child restraint instructions and the following steps: Example -- Rear Driver's Side Position 1-56 Example -- Rear Driver's Side Position 2.1. When using a child restraint with a top tether in the rear driver side position: A. Raise the headrest or head restraint. B. Route the top tether (B) between the headrest or head restraint posts, through the loop (A), behind the inboard headrest or head restraint post, and under the center shoulder belt (C). C. Attach the top tether (B) to the top tether anchor (loop) (D) at the center rear seating position. 2.2. When using a child restraint with a top tether in the rear center position: A. Route the top tether (B) through the center loop (D), and behind the inboard passenger side headrest or head restraint post. B. Attach the top tether (B) to the top tether anchor (loop) at the rear passenger side seating position. 2.3. When using a child restraint with a top tether in the rear passenger position: A. Raise the headrest or head restraint. B. Route the top tether (B) between the headrest or head restraint posts, through the loop on the passenger side and behind the inboard headrest or head restraint post. C. Attach the top tether (B) to the top tether anchor (loop) (D) at the center rear seating position. 2.4. Tighten the top tether when and as the child restraint manufacturer's instructions say. When the top tether is tightened, the anchor (loop) may bend. This is normal and will not damage the vehicle. 3. Push and pull the child restraint in different directions to be sure it is secure. 1-57 Securing a Child Restraint in a Rear Seat Position When securing a child restraint in a rear seating position, study the instructions that came with the child restraint to make sure it is compatible with this vehicle. If the child restraint has the LATCH system, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for how and where to install the child restraint using LATCH. If a child restraint is secured in the vehicle using a safety belt and it uses a top tether, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for top tether anchor locations. Do not secure a child seat in a position without a top tether anchor if a national or local law requires that the top tether be anchored, or if the instructions that come with the child restraint say that the top strap must be anchored. In Canada, the law requires that forward-facing child restraints have a top tether, and that the tether be attached. If the child restraint does not have the LATCH system, you will be using the safety belt to secure the child restraint in this position. Be sure to follow the instructions that came with the child restraint. Secure the child in the child restraint when and as the instructions say. If more than one child restraint needs to be installed in the rear seat, be sure to read Where to Put the Restraint on page 1-48. 1. Put the child restraint on the seat. 2. Pick up the latch plate, and run the lap and shoulder portions of the vehicle's safety belt through or around the restraint. The child restraint instructions will show you how. 1-58 For crew cab second row seatings positions, tilt the latch plate to adjust the belt if needed. 3. Push the latch plate into the buckle until it clicks. Position the release button on the buckle so that the safety belt could be quickly unbuckled if necessary. 1-59 4. Pull the rest of the shoulder belt all the way out of the retractor to set the lock. 5. To tighten the belt, push down on the child restraint, pull the shoulder portion of the belt to tighten the lap portion of the belt and feed the shoulder belt back into the retractor. When installing a forward-facing child restraint, it may be helpful to use your knee to push down on the child restraint as you tighten the belt. 1-60 6. If the child restraint has a top tether, follow the child restraint manufacturer's instructions regarding the use of the top tether. See Lower Anchors and Tethers for Children (LATCH) on page 1-49 for more information. 7. Push and pull the child restraint in different directions to be sure it is secure. To remove the child restraint, unbuckle the vehicle safety belt and let it return to the stowed position. If the top tether is attached to a top tether anchor, disconnect it. Securing a Child Restraint in the Center Front Seat Position { CAUTION: A child in a child restraint in the center front seat can be badly injured or killed by the frontal airbags if they inflate. Never secure a child restraint in the center front seat. It is always better to secure a child restraint in a rear seat. Do not use child restraints in the center front seat position. 1-61 Securing a Child Restraint in the Right Front Seat Position (With Airbag Off Switch) This vehicle has airbags. A rear seat is a safer place to secure a forward-facing child restraint. See Where to Put the Restraint on page 1-48. There may be a switch in the glove box that you can use to turn off the right front passenger frontal airbag. See Airbag Off Switch on page 1-81 for more information, including important safety information. A label on the sun visor says, "Never put a rear-facing child seat in the front." This is because the risk to the rear-facing child is so great, if the airbag deploys. { CAUTION: A child in a rear-facing child restraint can be seriously injured or killed if the right front passenger airbag inflates. This is because the back of the rear-facing child restraint would be very close to CAUTION: (Continued) CAUTION: (Continued) the inflating airbag. A child in a forward-facing child restraint can be seriously injured or killed if the right front passenger airbag inflates and the passenger seat is in a forward position. Even if the airbag switch has turned off the right front passenger frontal airbag, no system is fail-safe. No one can guarantee that an airbag will not deploy under some unusual circumstance, even though it is turned off. Secure rear-facing child restraints in a rear seat, even if the airbag is off. If you secure a forward-facing child restraint in the right front seat, always move the front passenger seat as far back as it will go. It is better to secure the child restraint in a rear seat. 1-62 { CAUTION: If the airbag readiness light ever comes on and stays on, it means that something may be wrong with the airbag system. For example, the right front passenger airbag could inflate even though the airbag on-off switch is turned off. To help avoid injury to yourself or others, have the vehicle serviced right away. See Airbag Readiness Light on page 3-37 for more information, including important safety information. If the vehicle does not have a rear seat that will accommodate a rear-facing child restraint, a rear-facing child restraint should not be installed in the vehicle, even if the airbag is off. If the child restraint has the LATCH system, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for how and where to install the child restraint using LATCH. If a child restraint is secured using a safety belt and it uses a top tether, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for top tether anchor locations. Do not secure a child restraint in a position without a top tether anchor if a national or local law requires that the top tether be anchored, or if the instructions that come with the child restraint say that the top strap must be anchored. In Canada, the law requires that forward-facing child restraints have a top tether, and that the tether be attached. You will be using the lap-shoulder belt to secure the child restraint in this position. Follow the instructions that came with the child restraint. 1. Move the seat as far back as it will go before securing the forward-facing child restraint. If you have no other choice but to install a rear-facing child restraint in this seat, make sure the airbag is off once the child restraint has been installed. When the airbag off switch has turned off the right front passenger frontal airbag, the off indicator in the airbag off light should light and stay lit when you start the vehicle. See Airbag Off Light on page 3-38. 2. Put the child restraint on the seat. 3. Pick up the latch plate, and run the lap and shoulder portions of the vehicle's safety belt through or around the restraint. The child restraint instructions will show you how. 1-63 4. Push the latch plate into the buckle until it clicks. Position the release button so that the safety belt could be quickly unbuckled if necessary. 5. Pull the rest of the shoulder belt all the way out of the retractor to set the lock. 1-64 6. To tighten the belt, push down on the child restraint, pull the shoulder portion of the belt to tighten the lap portion of the belt and feed the shoulder belt back into the retractor. When installing a forward-facing child restraint, it may be helpful to use your knee to push down on the child restraint as you tighten the belt. 7. If your vehicle does not have a rear seat and your child restraint has a top tether, follow the child restraint manufacturer's instructions regarding the use of the top tether. See Lower Anchors and Tethers for Children (LATCH) on page 1-49. 8. Push and pull the child restraint in different directions to be sure it is secure. To remove the child restraint, unbuckle the vehicle safety belt and let it return to the stowed position. If the top tether is attached to a top tether anchor, disconnect it. If you turned the airbag off with the switch, turn on the right front passenger airbag when you remove the child restraint from the vehicle unless the person who will be sitting there is a member of a passenger airbag risk group. See Airbag Off Switch on page 1-81. 1-65 Securing a Child Restraint in the Right Front Seat Position (With Passenger Sensing System) This vehicle has airbags. A rear seat is a safer place to secure a forward-facing child restraint. See Where to Put the Restraint on page 1-48. In addition, the vehicle may have a passenger sensing system which is designed to turn off the right front passenger frontal airbag under certain conditions. See Passenger Sensing System on page 1-84 and Passenger Airbag Status Indicator on page 3-40 for more information on this, including important safety information. A label on the sun visor says, "Never put a rear-facing child seat in the front." This is because the risk to the rear-facing child is so great, if the airbag deploys. 1-66 { CAUTION: A child in a rear-facing child restraint can be seriously injured or killed if the right front passenger airbag inflates. This is because the back of the rear-facing child restraint would be very close to the inflating airbag. A child in a forward-facing child restraint can be seriously injured or killed if the right front passenger airbag inflates and the passenger seat is in a forward position. Even if the passenger sensing system has turned off the right front passenger frontal airbag, no system is fail-safe. No one can guarantee that an airbag will not deploy under some unusual circumstance, even though it is turned off. Secure rear-facing child restraints in a rear seat, even if the airbag is off. If you secure a forward-facing child restraint in the right front seat, always move the front passenger seat as far back as it will go. It is better to secure the child restraint in a rear seat. See Passenger Sensing System on page 1-84 for additional information. If the vehicle does not have a rear seat that will accommodate a rear-facing child restraint, a rear-facing child restraint should not be installed in the vehicle, even if the airbag is off. If your child restraint has the LATCH system, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for how and where to install your child restraint using LATCH. If you secure a child restraint using a safety belt and it uses a top tether, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for top tether anchor locations. Do not secure a child seat in a position without a top tether anchor if a national or local law requires that the top tether be anchored, or if the instructions that come with the child restraint say that the top strap must be anchored. In Canada, the law requires that forward-facing child restraints have a top tether, and that the tether be attached. You will be using the lap-shoulder belt to secure the child restraint in this position. Follow the instructions that came with the child restraint. 1. Move the seat as far back as it will go before securing the forward-facing child restraint. When the passenger sensing system has turned off the right front passenger frontal airbag, the off indicator in the passenger airbag status indicator should light and stay lit when you start the vehicle. See Passenger Airbag Status Indicator on page 3-40. 2. Put the child restraint on the seat. 3. Pick up the latch plate, and run the lap and shoulder portions of the vehicle's safety belt through or around the restraint. The child restraint instructions will show you how. 1-67 4. Push the latch plate into the buckle until it clicks. Position the release button so that the safety belt could be quickly unbuckled if necessary. 5. Pull the rest of the shoulder belt all the way out of the retractor to set the lock. 1-68 6. To tighten the belt, push down on the child restraint, pull the shoulder portion of the belt to tighten the lap portion of the belt and feed the shoulder belt back into the retractor. When installing a forward-facing child restraint, it may be helpful to use your knee to push down on the child restraint as you tighten the belt. 7. If the vehicle does not have a rear seat and the child restraint has a top tether, follow the child restraint manufacturer's instructions regarding the use of the top tether. See Lower Anchors and Tethers for Children (LATCH) on page 1-49 for more information. 8. Push and pull the child restraint in different directions to be sure it is secure. If the airbag is off, the off indicator in the passenger airbag status indicator will come on and stay on when the vehicle is started. If a child restraint has been installed and the on indicator is lit, see "If the On Indicator is Lit for a Child Restraint" under Passenger Sensing System on page 1-84 for more information. To remove the child restraint, unbuckle the vehicle safety belt and let it return to the stowed position. If the top tether is attached to a top tether anchor, disconnect it. 1-69 Securing a Child Restraint in the Right Front Seat Position (Heavy Duty Crew Cab Only) Your vehicle has airbags. A rear seat is a safer place to secure a forward-facing child restraint. See Where to Put the Restraint on page 1-48. A label on the sun visor says, "Never put a rear-facing child seat in the front." This is because the risk to the rear-facing child is so great, if the airbag deploys. Never put a rear-facing child restraint in the right front passenger seat. Here is why: { CAUTION: A child in a rear-facing child restraint can be seriously injured or killed if the right front passenger's airbag inflates. This is because the back of the rear-facing child restraint would be very close to the inflating airbag. Always secure a rear-facing child restraint in a rear seat. If the vehicle does not have a rear seat that will accommodate a rear-facing child restraint, a rear-facing child restraint should not be installed in the vehicle, even if the airbag is off. If your child restraint has the LATCH system, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for how and where to install your child restraint using LATCH. If you secure a child restraint using a safety belt and it uses a top tether, see Lower Anchors and Tethers for Children (LATCH) on page 1-49 for top tether anchor locations. Do not secure a child seat in a position without a top tether anchor if a national or local law requires that the top tether be anchored, or if the instructions that come with the child restraint say that the top strap must be anchored. 1-70 In Canada, the law requires that forward-facing child restraints have a top tether, and that the tether be attached. You will be using the lap-shoulder belt to secure the child restraint in this position. Follow the instructions that came with the child restraint. 1. Move the seat as far back as it will go before securing the forward-facing child restraint. 2. Put the child restraint on the seat. 3. Pick up the latch plate, and run the lap and shoulder portions of the vehicle's safety belt through or around the restraint. The child restraint instructions will show you how. 4. Push the latch plate into the buckle until it clicks. Make sure the release button is positioned so you would be able to unbuckle the safety belt quickly if necessary. 1-71 5. Pull the rest of the shoulder belt all the way out of the retractor to set the lock. 6. To tighten the belt, push down on the child restraint, pull the shoulder portion of the belt to tighten the lap portion of the belt and feed the shoulder belt back into the retractor. If you are using a forward-facing child restraint, you may find it helpful to use your knee to push down on the child restraint as you tighten the belt. 1-72 7. If your child restraint has a top tether, follow the child restraint manufacturer's instructions regarding the use of the top tether. See Lower Anchors and Tethers for Children (LATCH) on page 1-49 for more information. 8. Push and pull the child restraint in different directions to be sure it is secure. To remove the child restraint, unbuckle the vehicle safety belt and let it return to the stowed position. If the top tether is attached to a top tether anchor, disconnect it. Airbag System The vehicle has the following airbags: · A frontal airbag for the driver. · A frontal airbag for the right front passenger. The vehicle may have the following airbags: · A roof-rail airbag for the driver and the passenger seated directly behind the driver. · A roof-rail airbag for the right front passenger and the person seated directly behind the right front passenger. All of the airbags in the vehicle will have the word AIRBAG embossed in the trim or on an attached label near the deployment opening. For frontal airbags, the word AIRBAG will appear on the middle part of the steering wheel for the driver and on the instrument panel for the right front passenger. With roof-rail airbags, the word AIRBAG will appear along the headliner or trim. Airbags are designed to supplement the protection provided by safety belts. Even though today's airbags are also designed to help reduce the risk of injury from the force of an inflating bag, all airbags must inflate very quickly to do their job. 1-73 Here are the most important things to know about the airbag system: { CAUTION: You can be severely injured or killed in a crash if you are not wearing your safety belt -- even if you have airbags. Airbags are designed to work with safety belts, but do not replace them. Also, airbags are not designed to deploy in every crash. In some crashes safety belts are your only restraint. See When Should an Airbag Inflate? on page 1-77. Wearing your safety belt during a crash helps reduce your chance of hitting things inside the vehicle or being ejected from it. Airbags are "supplemental restraints" to the safety belts. Everyone in your vehicle should wear a safety belt properly -- whether or not there is an airbag for that person. { CAUTION: Airbags inflate with great force, faster than the blink of an eye. Anyone who is up against, or very close to, any airbag when it inflates can be seriously injured or killed. Do not sit unnecessarily close to the airbag, as you would be if you were sitting on the edge of your seat or leaning forward. Safety belts help keep you in position before and during a crash. Always wear your safety belt, even with airbags. The driver should sit as far back as possible while still maintaining control of the vehicle. Occupants should not lean on or sleep against the door or side windows in seating positions with roof-rail airbags. 1-74 { CAUTION: Children who are up against, or very close to, any airbag when it inflates can be seriously injured or killed. Airbags plus lap-shoulder belts offer protection for adults and older children, but not for young children and infants. Neither the vehicle's safety belt system nor its airbag system is designed for them. Young children and infants need the protection that a child restraint system can provide. Always secure children properly in your vehicle. To read how, see Older Children on page 1-38 or Infants and Young Children on page 1-41. There is an airbag readiness light on the instrument panel cluster, which shows the airbag symbol. Where Are the Airbags? The driver's airbag is in the middle of the steering wheel. The system checks the airbag electrical system for malfunctions. The light tells you if there is an electrical problem. See Airbag Readiness Light on page 3-37 for more information. 1-75 The right front passenger's airbag is in the instrument panel on the passenger's side. Driver Side shown, Passenger Side similar If your vehicle has roof-rail airbags for the driver, right front passenger, and second row outboard passengers, they are in the ceiling above the side windows. 1-76 { CAUTION: If something is between an occupant and an airbag, the airbag might not inflate properly or it might force the object into that person causing severe injury or even death. The path of an inflating airbag must be kept clear. Do not put anything between an occupant and an airbag, and do not attach or put anything on the steering wheel hub or on or near any other airbag covering. Never secure anything to the roof of a vehicle with roof-rail airbags by routing a rope or tie down through any door or window opening. If you do, the path of an inflating roof-rail airbag will be blocked. When Should an Airbag Inflate? Frontal airbags are designed to inflate in moderate to severe frontal or near-frontal crashes to help reduce the potential for severe injuries mainly to the driver's or right front passenger's head and chest. However, they are only designed to inflate if the impact exceeds a predetermined deployment threshold. Deployment thresholds are used to predict how severe a crash is likely to be in time for the airbags to inflate and help restrain the occupants. Whether the frontal airbags will or should deploy is not based on how fast your vehicle is traveling. It depends largely on what you hit, the direction of the impact, and how quickly your vehicle slows down. 1-77 Frontal airbags may inflate at different crash speeds. For example: · If the vehicle hits a stationary object, the airbags could inflate at a different crash speed than if the vehicle hits a moving object. · If the vehicle hits an object that deforms, the airbags could inflate at a different crash speed than if the vehicle hits an object that does not deform. · If the vehicle hits a narrow object (like a pole), the airbags could inflate at a different crash speed than if the vehicle hits a wide object (like a wall). · If the vehicle goes into an object at an angle, the airbags could inflate at a different crash speed than if the vehicle goes straight into the object. Thresholds can also vary with specific vehicle design. Frontal airbags are not intended to inflate during vehicle rollovers, rear impacts, or in many side impacts. If the GVWR (Gross Vehicle Weight Rating) of your vehicle is 8,500 lb (3 855 kg) or above, your vehicle has single stage airbags. If the GVWR is below 8,500 lb (3 855 kg) then your vehicle has dual stage airbags. You can find the GVWR on the certification label on the rear edge of the driver's door. See Loading the Vehicle on page 4-32 for more information. In addition, the vehicle may have dual-stage frontal airbags. Dual-stage airbags adjust the restraint according to crash severity. The vehicle has electronic frontal sensors, which help the sensing system distinguish between a moderate frontal impact and a more severe frontal impact. For moderate frontal impacts, dual-stage airbags inflate at a level less than full deployment. For more severe frontal impacts, full deployment occurs. Vehicles with dual stage airbags also have seat position sensors which enable the sensing system to monitor the position of the driver seat (all models) and the right front passenger seat (light-duty regular cab and light-duty extended cab models only). The seat position sensor provides information that is used to determine if the airbags should deploy at a reduced level or at full deployment. The vehicle may or may not have roof-rail airbags. See Airbag System on page 1-73. Roof-rail airbags are intended to inflate in moderate to severe side crashes. In addition, these roof-rail airbags are intended to inflate during a rollover or in a severe frontal impact. Roof-rail airbags will inflate if the crash severity is above the system's designed threshold level. The threshold level can vary with specific vehicle design. 1-78 Roof-rail airbags are not intended to inflate in rear impacts. Both roof-rail airbags will deploy when either side of the vehicle is struck, or if the sensing system predicts that the vehicle is about to roll over, or in a severe frontal impact. In any particular crash, no one can say whether an airbag should have inflated simply because of the damage to a vehicle or because of what the repair costs were. For frontal airbags, inflation is determined by what the vehicle hits, the angle of the impact, and how quickly the vehicle slows down. For roof-rail airbags, deployment is determined by the location and severity of the side impact. In a rollover event, roof-rail airbag deployment is determined by the direction of the roll. What Makes an Airbag Inflate? In a deployment event, the sensing system sends an electrical signal triggering a release of gas from the inflator. Gas from the inflator fills the airbag causing the ba etting the Clock on page 3-86 for more information. Speed Compensated Volume (SCV): Radios with Speed Compensated Volume (SCV) automatically adjusts the radio volume to compensate for road and wind noise as the vehicle's speed changes while driving, so that the volume level stays consistent. To activate SCV: 1. Set the radio volume to the desired level. 2. Press the MENU button to display the radio setup menu. 3. Press the pushbutton under the AUTO VOLUM (automatic volume) label on the radio display. 4. Press the pushbutton under the desired Speed Compensated Volume setting (OFF, Low, Med, or High) to select the level of radio volume compensation. The display times out after approximately 10 seconds. Each higher setting allows for more radio volume compensation at faster vehicle speeds. Finding a Station BAND: Press to switch between AM, FM, or XMTM (if equipped). The selection displays. f (Tune): Turn to select radio stations. ©SEEK ¨: Press either arrow to go to the previous or to the next station and stay there. To scan stations, press and hold either arrow for a few seconds until a beep sounds. The radio goes to a station, plays for a few seconds, then goes to the next station. For AM-FM Radio, the station frequency flashes while the radio is in the scan mode. Press either arrow again to stop scanning. The radio only seeks and scans stations with a strong signal that are in the selected band. For the AM-FM Radio, scan presets within the current selected band by pressing and holding either seek arrow for four seconds until a double beep sounds. The radio goes to a stored preset, plays for a few seconds if a strong signal is present, then goes to the next stored preset. The station frequency flashes while the radio is in the scan mode. 3-91 4 (Information) (XM Satellite Radio Service, MP3, and RDS Features): Press to switch the display between the radio station frequency and the time. When the ignition is in the OFF position, press 4 to display the time. For vehicles with XM, MP3, WMA or RDS features, press 4 to display additional text information related to the current FM-RDS or XM station; or CD, MP3 or WMA song. If information is available during XM, CD, MP3 or WMA playback, the song title information displays on the top line of the display and artist information displays on the bottom line. When information is not available, "NO INFO" displays. Setting Preset Stations (AM-FM Radio) If the radio does not have XM, up to 18 stations (six FM1, six FM2, and six AM), can be programmed on the six numbered pushbuttons. To program presets: 1. Turn the radio on. 2. Press BAND to select FM1, FM2, or AM. 3. Tune in the desired station. 4. Press and hold one of the six numbered pushbuttons for three seconds until a beep sounds. Whenever that pushbutton is pressed and released, the station that was set, returns. 5. Repeat the Steps 2 through 4 for each pushbutton. 3-92 Storing a Radio Station as a Favorite Drivers are encouraged to set up their radio station favorites while the vehicle is parked. Tune to favorite stations using the presets, favorites button, and steering wheel controls, if the vehicle has this feature. See Defensive Driving on page 4-2. FAV (Favorites): If the vehicle has XM and has a FAV button, a maximum of 36 stations can be programmed as favorites using the six pushbuttons positioned below the radio station frequency labels and by using the radio favorites page button (FAV button). Press the FAV button to go through up to six pages of favorites, each having six favorite stations available per page. Each page of favorites can contain any combination of AM, FM, or XM (if equipped) stations. The balance/fade and tone settings that were previously adjusted, are stored with the favorite stations. To store a station as a favorite: 1. Tune to the desired radio station. 2. Press the FAV button to display the page where the station will be stored. 3. Press and hold one of the six pushbuttons until a beep sounds. When that pushbutton is pressed and released, the station that was set, returns. 4. Repeat the steps for each pushbutton radio station to store as a favorite. The number of favorites pages can be setup using the MENU button. To setup the number of favorites pages: 1. Press the MENU button to display the radio setup menu. 2. Press the pushbutton located below the FAV 1-6 label. 3. Select the desired number of favorites pages by pressing the pushbutton located below the displayed page numbers. 4. Press the FAV button, or let the menu time out, to return to the original main radio screen showing the radio station frequency labels and to begin the process of programming favorites for the chosen amount of numbered pages. Setting the Tone (Bass/Treble) (AM-FM Radio) Bass/Treble: To adjust the bass or treble, press the tune knob or the EQ button until the desired tone control label displays. Turn the tune knob clockwise or counterclockwise to increase or decrease the setting. The display shows the current bass or treble level. If a station's frequency is weak, or if there is static, decrease the treble. Setting the Tone (Bass/Midrange/Treble) BASS/MID/TREB (Bass, Midrange, or Treble): To adjust bass, midrange, or treble, press the f knob until the tone control labels display. Continue pressing to highlight the desired label, or press the pushbutton positioned under the desired label. Turn the f knob clockwise or counterclockwise to adjust the highlighted setting, or adjust the highlighted setting by pressing either SEEK arrow, \ FWD (forward), or s REV (reverse) button until the desired levels are obtained. If a station's frequency is weak or if there is static, decrease the treble. To quickly adjust bass, midrange, or treble to the middle position, press the pushbutton positioned under the BASS, MID, or TREB label for more than two seconds. A beep sounds and the level adjusts to the middle position. To quickly adjust all tone and speaker controls to the middle position, press the f knob for more than two seconds until a beep sounds. 3-93 EQ (Equalization): Press this button to choose bass and treble equalization settings designed for different types of music. The choices are pop, rock, country, talk, jazz, and classical. Selecting MANUAL or changing bass or treble, returns the EQ to the manual bass and treble settings. Unique EQ settings can be saved for each source. If the radio has a Bose® audio system, the EQ settings are either MANUAL or TALK. Adjusting the Speakers (Balance/Fade) (AM-FM Radio) ` (Balance/Fade): To adjust the balance or fade, press this button or the tune knob until the desired speaker control label displays. Turn the tune knob clockwise or counterclockwise to adjust the setting. The setting can also be adjusted by pressing the seek arrows. Adjusting the Speakers (Balance/Fade) BAL/FADE (Balance/Fade): To adjust balance or fade, press the f knob until the speaker control labels display. Continue pressing to highlight the desired label, or press the pushbutton positioned under the desired label. Turn the f knob clockwise or counterclockwise to adjust the highlighted setting, or adjust the highlighted setting by pressing either SEEK arrow, \ FWD, or s REV button until the desired levels are obtained. To quickly adjust balance or fade to the middle position, press the pushbutton positioned under the BAL or FADE label for more than two seconds. A beep sounds and the level adjusts to the middle position. To quickly adjust all speaker and tone controls to the middle position, press the f knob for more than two seconds. If the Rear Seat Audio (RSA) is turned on, the radio disables FADE and mutes the rear speakers. 3-94 Finding a Category (CAT) Station CAT (Category): The CAT button is used to find XM stations when the radio is in the XM mode. To find XM channels within a desired category: 1. Press the BAND button until the XM frequency displays. Press the CAT button to display the category labels. Continue pressing the CAT button until the desired category name displays. · Radios with CD and DVD can also navigate the category list by pressing the \ FWD or the s REV button. 2. Press either of the two buttons below the desired category label to immediately tune to the first XM station associated with that category. 3. Turn the f knob, press the buttons below the right or left arrows displayed, or press either SEEK arrow to go to the previous or to the next XM station within the selected category. 4. To exit the category search mode, press the FAV button or BAND button to display the favorites again. Undesired XM categories can be removed through the setup menu. To remove an undesired category: 1. Press the MENU button to display the radio setup menu. 2. Press the pushbutton located below the XM CAT label. 3. Turn the f knob to display the category to be removed. 4. Press the pushbutton located under the Remove label until the category name along with the word Removed displays. 5. Repeat the steps to remove more categories. Removed categories can be restored by pressing the pushbutton under the Add label when a removed category is displayed or by pressing the pushbutton under the Restore All label. Categories cannot be removed or added while the vehicle is moving faster than 5 mph (8 km/h). 3-95 Radio Messages Calibration Error: The audio system has been calibrated for the vehicle from the factory. If Calibration Error displays, it means that the radio has not been configured properly for the vehicle and it must be returned to your dealer/retailer for service. Locked or Loc: One of these messages will display when the THEFTLOCK® system has locked up the radio. Take the vehicle to your dealer/retailer for service. If any error occurs repeatedly or if an error cannot be corrected, contact your dealer/retailer. XMTM Satellite Radio Service XM is a satellite radio service that is based in the 48 contiguous United States and 10 Canadian provinces. XM Satellite Radio has a wide variety of programming and commercial-free music, coast-to-coast, and in digital-quality sound. During your trial or when you subscribe, you will get unlimited access to XM Radio Online for when you are not in the vehicle. A service fee is required to receive the XM service. For more information, contact XM at xmradio.com or call 1-800-929-2100 in the U.S. and xmradio.ca or call 1-877-438-9677 in Canada. Radio Messages for XM Only See XM Radio Messages on page 3-117 later in this section for further detail. 3-96 Playing a CD (Single CD Player) Insert a CD partway into the slot, label side up. The player pulls it in and the CD should begin playing. Playing a CD(s) (Six-Disc CD Player) LOAD ^ : Press to load CDs into the CD player. This CD player holds up to six CDs. To insert one CD: 1. Press and release the ^ button. 2. Wait for the message to insert the disc. 3. Load a CD. Insert the CD partway into the slot, label side up. The player pulls the CD in. To insert multiple CDs: 1. Press and hold the ^ button for two seconds. A beep sounds and Load All Discs displays. 2. Follow the displayed instruction on when to insert the discs. The CD player takes up to six CDs. 3. Press the ^ button again to cancel loading more CDs. Insert a CD partway into the slot, label side up. The player pulls it in and the CD should begin playing. To insert a CD with the ignition off, first press the Z button or the DISP knob. If the ignition or radio is turned off with a CD in the player it stays in the player. When the ignition or radio is turned on, the CD starts to play where it stopped, if it was the last selected audio source. When the CD is inserted, the CD symbol displays. As each new track starts to play, the track number displays. The CD player can play the smaller 3 inch (8 cm) single CDs with an adapter ring. Full-size CDs and the smaller CDs are loaded in the same manner. Playing a CD (In Either the DVD or CD Slot) Insert a CD partway into the slot, label side up. The player pulls it in and the CD should begin playing (loading a disc into the system, depending on media type and format ranges from 5 to 20 seconds for a CD, and up to 30 seconds for a DVD to begin playing). If the ignition or radio is turned off, with a CD in the player, it stays in the player. When the ignition or radio is turned on, the CD starts playing where it stopped, if it was the last selected audio source. The CD is controlled by the buttons on the radio faceplate or by the RSA unit. See Rear Seat Audio (RSA) on page 3-138 for more information. The DVD/CD decks, (upper slot is the DVD deck and the lower slot is the CD deck) of the radio are compatible with most audio CDs, CD-R, CD-RW, and MP3s. When a CD is inserted, the text label DVD or CD symbol displays on the left side of the radio display. As each new track starts to play, the track number displays. The CD player can play the smaller 3 inch (8 cm) single CDs with an adapter ring. Full-size CDs and the smaller CDs are loaded in the same manner. Care of CDs If playing a CD-R, the sound quality can be reduced due to CD-R or CD-RW quality, the method of recording, the quality of the music that has been recorded, and the way the CD-R or CD-RW has been handled. Handle them carefully. Store CD-R(s) or CD-RW(s) in their original cases or other protective cases and away from direct sunlight and dust. The CD or DVD player scans the bottom surface of the disc. If the surface of a CD is damaged, such as cracked, broken, or scratched, the CD does not play properly or not at all. Do not touch the bottom side of a CD while handling it; this could damage the surface. Pick up CDs by grasping the outer edges or the edge of the hole and the outer edge. If the surface of a CD is soiled, take a soft, lint free cloth or dampen a clean, soft cloth in a mild, neutral detergent solution mixed with water, and clean it. Make sure the wiping process starts from the center to the edge. 3-97 Care of the CD and DVD Player Do not add any label to a CD, it could get caught in the CD or DVD player. If a CD is recorded on a personal computer and a description label is needed, try labeling the top of the recorded CD with a marking pen instead. The use of CD lens cleaners for CDs is not advised, due to the risk of contaminating the lens of the CD optics with lubricants internal to the CD and DVD player mechanism. Notice: If a label is added to a CD, or more than one CD is inserted into the slot at a time, or an attempt is made to play scratched or damaged CDs, the CD player could be damaged. While using the CD player, use only CDs in good condition without any label, load one CD at a time, and keep the CD player and the loading slot free of foreign materials, liquids, and debris. If an error displays, see "CD Messages" later in this section. Z EJECT or CD (Eject): Press and release to eject the disc that is currently playing. A CD ejecting from a radio with CD and DVD, ejects from the bottom slot. A beep sounds and Ejecting Disc displays. Once the disc is ejected, Remove Disc displays. The disc can be removed. If the disc is not removed, after several seconds, the disc automatically pulls back into the player. For the Six-Disc CD player, press and hold for two seconds to eject all discs. 3-98 Z DVD (Eject): Press and release to eject the disc that is currently playing in the top slot. A beep sounds and Ejecting Disc displays. If loading and reading of a disc cannot be completed, such as unknown format, etc., and the disc fails to eject, press and hold for more than five seconds to force the disc to eject. f (Tune): Turn to select tracks on the CD that is currently playing. ©SEEK ¨: Press the left arrow to go to the start of the current track, if more than ten seconds on the CD have played. Press the right arrow to go to the next track. For Radios with CD and DVD, Press the left arrow to go to the start of the current track, if more than five seconds on the CD have played. If less than five seconds on the CD have played, the previous track plays. Press the right arrow to go to the next track. If either arrow is held, or pressed multiple times, the player continues moving backward or forward through the tracks on the CD. s REV (Fast Reverse): Press and hold to reverse playback quickly within a track. Sound will be heard at a reduced volume. Release to resume playing the track. The elapsed time of the track displays. \ FWD (Fast Forward): Press and hold to advance playback quickly within a track. Sound will be heard at a reduced volume. Release to resume playing the track. The elapsed time of the track displays. RDM (Random): With the random setting, the tracks can be listened to in random, rather than sequential order. To use random, do one of the following: · Press the CD/AUX button, or for a single CD player, insert a disc partway into the slot of the CD player. A RDM label displays. To play the tracks from the single CD in random order, press the pushbutton positioned under the RDM label until Random Current Disc displays. Press the pushbutton again to turn off random play. · Press the CD/AUX button, or for a six-disc CD player, press and hold the ^ button. A beep sounds and Load All Discs displays. Insert one or more discs partway into the slot of the CD player. To play tracks from all CDs loaded in a six-disc CD player in random order, press the pushbutton positioned under the RDM label until Randomize All Discs displays. Press the same pushbutton again to turn off random play. To play the tracks from a CD loaded in the radio with CD and DVD, press the DVD/CD AUX button when not sourced to the CD, or insert a disc partway into the slot. A RDM label displays. To play tracks from a single CD in random order, press the pushbutton positioned under the RDM label until Random Current Disc displays. Press the pushbutton again to turn off random play. BAND: Press to listen to the radio when a CD is playing. The CD remains inside the radio for future listening. For the radio with CD and DVD, press to listen to the radio when a CD or DVD is playing. The CD or DVD remains inside the radio for future listening or for viewing entertainment. CD/AUX (CD/Auxiliary): Press to play a CD when listening to the radio. The CD icon and a message showing the disc and/or track number displays when a CD is in the player. Press again and the system automatically searches for an auxiliary input device, such as a portable audio player. If a portable audio player is not connected, "No Input Device Found" displays. 3-99 DVD/CD AUX (Auxiliary): Press this button to cycle through DVD, CD, or Auxiliary when listening to the radio. The DVD/CD text label and a message showing the track or chapter number displays when a disc is in either slot. Press this button again and the system automatically searches for an auxiliary input device, such as a portable audio player. If a portable audio player is not connected, "No Aux Input Device" displays. If a disc is in both the DVD slot and the CD slot the DVD/CD AUX button cycles between the two sources and not indicate "No Aux Input Device". If a front auxiliary device is connected, the DVD/CD AUX button cycles through all available options, such as: DVD slot, CD slot, Front Auxiliary, and Rear Auxiliary (if available). See "Using the Auxiliary Input Jack(s)" later in this section, or "Audio/Video (A/V) Jacks" under, Rear Seat Entertainment (RSE) System on page 3-129 for more information. If a disc is inserted into top DVD slot, the rear seat operator can turn on the video screen and use the remote control to navigate the CD (tracks only) through the remote control. Radios with CD and DVD Audio Output Only one audio source can be heard through the speakers at one time. An audio source is defined as DVD slot, CD slot, XM, FM/AM, Front Auxiliary Jack, or Rear Auxiliary Jack. Press the O button to turn the radio on. The radio can be heard through all of the vehicle speakers. Front seat passengers can listen to the radio (AM, FM, or XM) by pressing the BAND button or the DVD/CD AUX button to select CD slot, DVD slot, front or rear auxiliary input (if available). If a playback device is plugged into the radio's front auxiliary input jack or the rear auxiliary jack, the front seat passengers are able to listen to playback from this source through the vehicle speakers. See "Using the Auxiliary Input Jack(s)" later in this section, or "Audio/Video (A/V) Jacks" under, Rear Seat Entertainment (RSE) System on page 3-129 for more information. In some vehicles, depending on audio options, the rear speakers can be muted when the RSA power is turned on. See Rear Seat Audio (RSA) on page 3-138 for more information. 3-100 Playing an MP3 CD-R or CD-RW Disc Radios with a Single CD player or a Six-Disc CD player has the capability of playing an MP3 CD-R or CD-RW disc. For more information on how to play an MP3 CD-R or CD-RW disc, see "Using an MP3" in the index. Playing an MP3/WMA CD-R or CD-RW Disc Radios with CD and DVD has the capability of playing an MP3/WMA CD-R or CD-RW disc. For more information on how to play an MP3/WMA CD-R or CD-RW disc, see "Using an MP3" in the index. CD Messages CHECK DISC: Radios with a Single CD player or radios with a Six-Disc player displays CHECK DISC and/or ejects the CD if an error occurs. Radios with a CD and DVD player may display other messages when an error occurs: Optical Error: The disc was inserted upside down. Disk Read Error: A disc was inserted with an invalid or unknown format. Player Error: There are disc LOAD or disc EJECT problems. · It is very hot. When the temperature returns to normal, the CD should play. · The road is very rough. When the road becomes smoother, the CD should play. · The CD is dirty, scratched, wet, or upside down. · The air is very humid. If so, wait about an hour and try again. · There could have been a problem while burning the CD. · The label could be caught in the CD player. If the CD is not playing correctly, for any other reason, try a known good CD. Radios with a CD and DVD player displays If any error occurs repeatedly or if an error cannot be corrected, contact your dealer/retailer. If the radio displays an error message, write it down and provide it to your dealer/retailer when reporting the problem. 3-101 Using the DVD Player The DVD player is controlled by the buttons on the remote control, or by the RSA system, or by the buttons on the radio faceplate. See "Remote Control", under Rear Seat Entertainment (RSE) System on page 3-129 and Rear Seat Audio (RSA) on page 3-138 for more information. The DVD player is only compatible with DVDs of the appropriate region code that is printed on the jacket of most DVDs. The DVD slot of the radio is compatible with most audio CDs, CD-R, CD-RW, DVD-Video, DVD-Audio, DVD-R/RW, DVD+R/RW media along with MP3 and WMA formats. If an error message displays on the video screen or the radio, see "DVD Display Error Messages" under, Rear Seat Entertainment (RSE) System on page 3-129 and "DVD Radio Error Messages" in this section for more information. Playing a DVD DVD/CD AUX (Auxiliary): Press this button to cycle through DVD, CD, or Auxiliary when listening to the radio. The DVD/CD text label and a message showing track or chapter number displays when a disc is in either slot. Press this button again and the system automatically searches for an auxiliary input device, such as a portable audio player. If a portable audio player is not connected, "No Aux Input Device" displays. If a disc is in both the DVD slot and the CD slot the DVD/CD AUX button cycles between the two sources and not indicate "No Aux Input Device". If a front auxiliary device is connected, the DVD/CD AUX button cycles through all available options, such as: DVD slot, CD slot, Front Auxiliary, and Rear Auxiliary (if available). See "Using the Auxiliary Input Jack(s)" later in this section, or "Audio/Video (A/V) Jacks" under, Rear Seat Entertainment (RSE) System on page 3-129 for more information. O (Power): Press to turn the radio on or off. Turn this knob clockwise or counterclockwise to increase or decrease the volume. Press and hold for more than two seconds to turn off the entire radio and Rear Seat Entertainment (RSE) system and to start the parental 3-102 control feature. Parental control prevents the rear seat occupant from operating the Rear Seat Audio (RSA) system or remote control. A lock symbol displays next to the clock display. The parental control feature remains on until the knob is pressed and held for more than two seconds again, or until the driver turns the ignition off and exits the vehicle. f (Tune): Turn to change tracks on a CD or DVD, to manually tune a radio station, or to change clock or date settings, while in the clock or date setting mode. See the information given earlier in this section specific to the radio, CD, and the DVD. Also, see "Setting the Time" in the index, for setting the clock and date. ©SEEK (Previous Track/Chapter): Press the left arrow to return to the start of the current track or chapter. Press the left arrow again to go to the previous track or chapter. This button might not work when the DVD is playing the copyright information or the previews. SEEK ¨(Next Track/Chapter): Press the right arrow to go to the next track or chapter. This button might not work when the DVD is playing the copyright information or the previews. s REV (Fast Reverse): Press to quickly reverse the CD or DVD at five times the normal speed. The radio displays the elapsed time while in fast reverse. To stop fast reversing, press again. This button might not work when the DVD is playing the copyright information or the previews. \ FWD (Fast Forward): Press to fast forward the CD or DVD. The radio displays the elapsed time and fast forwards five times the normal speed. To stop fast forwarding, press again. This button might not work when the DVD is playing the copyright information or the previews. Z (Eject): Press to eject a CD or DVD. If a CD or DVD is ejected, but not removed, the player automatically pulls it back in after 15 seconds. If loading and reading of a CD cannot be completed, because of an unknown format, etc., and the disc fails to eject, press and hold for more than five seconds to force the disc to eject. 3-103 DVD-V (Video) Display Buttons Once a DVD-V is inserted, the radio display menu shows several tag options for DVD playing. Press the pushbuttons located under any desired tag option during DVD playback. See the tag options listed after, for more information. The rear seat passenger can navigate the DVD-V menus and controls through the remote control. See "Remote Control", under Rear Seat Entertainment (RSE) System on page 3-129 for more information. The Video Screen automatically turns on when the DVD-V is inserted into the DVD slot. r / j (Play/Pause): Press either the play or pause icon displayed on the radio system, to toggle between pausing or restarting playback of a DVD. If the forward arrow is showing on display, the system is in pause mode. If the pause icon is showing on display, the system is in playback mode. If the DVD screen is off, press the play button to turn the screen on. Some DVDs begin playing after the previews have finished, although there could be a delay of up to 30 seconds. If the DVD does not begin playing the movie automatically, press the pushbutton located under the play/pause symbol tag displayed on the radio. If the DVD still does not play, refer to the on-screen instructions, if available. c (Stop): Press to stop playing, rewinding, or fast forwarding a DVD. r (Enter): Press to select the choices that are highlighted in any menu. y (Menu): Press to access the DVD menu. The DVD menu is different on every DVD. Use the pushbuttons located under the navigation arrows to navigate the cursor through the DVD menu. After making a selection press this button. This button only operates when using a DVD. Nav (Navigate): Press to display directional arrows for navigating through the menus. q (Return): Press to exit the current active menu and return to the previous menu. This button operates only when a DVD is playing and a menu is active. 3-104 DVD-A (Audio) Display Buttons Once a DVD-A is inserted, radio display menu shows several tag options for DVD playing. Press the pushbuttons located under any desired tag option during DVD playback. See the tag options listed after, for more information. The rear seat operator can navigate the DVD-A menus and controls through the remote control. See "Remote Control", under Rear Seat Entertainment (RSE) System on page 3-129 for more information. The Video Screen does not automatically power on when the DVD-A is inserted into the DVD slot. It must be manually turned on by the rear seat occupant through the remote control power button. r / j (Play/Pause): Press either the play or pause icon displayed on the radio system, to toggle between pausing or restarting playback of a DVD. If the forward arrow is showing on the display, the system is in pause mode. If the pause icon is showing on the display, the system is in playback mode. q Group r: Press to cycle through musical groupings on the DVD-A disc. Nav (Navigate): Press to display directional arrows for navigating through the menus. e (Audio Stream): Press to cycle through audio stream formats located on the DVD-A disc. The video screen shows the audio stream changing. Inserting a Disc To play a disc, gently insert the disc, with the label side up, into the loading slot. The DVD player might not accept some paper labeled media. The player starts loading the disc into the system and show "Loading Disc" on the radio display. At the same time, the radio displays a softkey menu of option(s). Some discs automatically play the movie while others default to the softkey menu display, which requires the Play, Enter, or Navigation softkeys to be pressed; either by softkey or by the rear seat passenger using the remote control. Loading a disc into the system, depending on media type and format, ranges from 5 to 20 seconds for a CD, and up to 30 seconds for a DVD. 3-105 Stopping and Resuming Playback To stop playing a DVD without turning off the system, press the c button on the remote control, or press the pushbutton located under the stop or the play/pause symbol tags displayed on the radio. If the radio head is sourced to something other than DVD-V, press the DVD/CD AUX button to make DVD-V the active source. To resume DVD playback, press the r / j button on the remote control, or press the pushbutton located under the play/pause symbol tag displayed on the radio. The DVD should resume play from where it last stopped if the disc has not been ejected and the stop button has not been pressed twice on the remote control. If the disc has been ejected or the stop button has been pressed twice on the remote control, the disc resumes playing at the beginning of the disc. Ejecting a Disc Press the Z button on the radio to eject the disc. If a disc is ejected from the radio, but not removed, the radio reloads the disc after a short period of time. The disc is stored in the radio. The radio does not resume play of the disc automatically. If the RSA system is sourced to the DVD, the movie when reloaded into the DVD player begins to play again. In case loading and reading of a DVD or CD cannot be completed (unknown format, etc.), and the disc fails to eject, press and hold the DVD Z button more than five seconds to force the disc to eject. DVD Radio Error Messages Player Error: This message displays when there are disc load or eject problems. Disc Format Error: This message displays, if the disc is inserted with the disc label wrong side up, or if the disc is damaged. Disc Region Error: This message displays, if the disc is not from a correct region. No Disc Inserted: This message displays, if no disc is present when the Z or DVD/CD AUX button is pressed on the radio. 3-106 Using the Auxiliary Input Jack The radio system has an auxiliary input jack located on the lower right side of the faceplate. This is not an audio output; do not plug the headphone set into the front auxiliary input jack. An external audio device such as an iPod, laptop computer, MP3 player, CD player, etc. can be connected to the auxiliary input jack for use as another source for audio listening. Drivers are encouraged to set up any auxiliary device while the vehicle is in P (Park). See Defensive Driving on page 4-2 for more information on driver distraction. To use a portable audio player, connect a 3.5 mm (1/8 inch) cable to the radio's front auxiliary input jack. When a device is connected, press the radio CD/AUX button to begin playing audio from the device over the vehicle speakers. For optimal sound quality, increase the portable audio device's volume to the loudest level. It is always best to power the portable audio device through its own battery while playing. O (Power/Volume): Turn clockwise or counterclockwise to increase or decrease the volume of the portable player. Additional volume adjustments might have to be made from the portable device if the volume is not loud or soft enough. BAND: Press to listen to the radio when a portable audio device is playing. The portable audio device continues playing. CD/AUX (CD/Auxiliary): Press to play a CD when a portable audio device is playing. Press again and the system begins playing audio from the connected portable audio player. If a portable audio player is not connected, "No Input Device Found" displays. DVD/CD AUX (CD/Auxiliary): Press to cycle through DVD, CD, or Auxiliary when listening to the radio. The DVD/CD text label and a message showing track or chapter number displays when a disc is in either slot. Press again and the system automatically searches for an auxiliary input device, such as a portable audio player. If a portable audio player is not connected, "No Aux Input Device" displays. If a disc is in both the DVD slot and the CD slot the DVD/CD AUX button cycles between the two sources and not indicate "No Aux Input Device". If a front auxiliary device is connected, the DVD/CD AUX button cycles through all available options, such as: DVD slot, CD slot, Front Auxiliary, and Rear Auxiliary (if available). See "Using the Auxiliary Input Jack(s)" later in this section, or "Audio/Video (A/V) Jacks" under, Rear Seat Entertainment (RSE) System on page 3-129 for more information. 3-107 Using an MP3 (Radio with CD or Six-Disc CD Player) MP3 CD-R or CD-RW Disc The radio plays MP3 files that were recorded on a CD-R or CD-RW disc. The files can be recorded with the following fixed bit rates: 32 kbps, 40 kbps, 56 kbps, 64 kbps, 80 kbps, 96 kbps, 112 kbps, 128 kbps, 160 kbps, 192 kbps, 224 kbps, 256 kbps, and 320 kbps or a variable bit rate. Song title, artist name, and album are available for display by the radio when recorded using ID3 tags version 1 and 2. Compressed Audio The radio also plays discs that contain both uncompressed CD audio (.CDA files) and MP3 files. By default the radio shows the MP3 label on the left side of the screen but plays both file formats in the order in which they were recorded to the disc. MP3 Format Burning an MP3 disc on a personal computer: · Make sure the MP3 files are recorded on a CD-R or CD-RW disc. · Do not mix standard audio and MP3 files on one disc. 3-108 · Make sure the CD does not have more than a maximum of 50 folders, 15 playlists, and 512 folders and files to read and play. · Create a folder structure that makes it easy to find songs while driving. Organize songs by albums using one folder for each album. Each folder or album should contain 18 songs or less. · Avoid subfolders. The system can support up to eight subfolders deep, however, keep the total number of folders to a minimum in order to reduce the complexity and confusion in trying to locate a particular folder during playback. · Make sure playlists have a .m3u extension as other file extensions might not work. · Minimize the length of the file, folder, or playlist names. Long file, folder, or playlist names, or a combination of a large number of files and folders, or playlists could cause the player to be unable to play up to the maximum number of files, folders, playlists, or sessions. To play a large number of files, folders, playlists or sessions, minimize the length of the file, folder, or playlist name. Long names also take up more space on the display, potentially getting cut off. · Finalize the audio disc before burning it. Trying to add music to an existing disc could cause the disc not to function in the player. Playlists can be changed by using the S c (previous) and c T (next) folder buttons, the f knob, or the ©SEEK ¨ arrows. MP3 CD-R or CD-RW that have been recorded without using file folders can be played. If a CD-R or CD-RW contains more than the maximum of 50 folders, 15 playlists, and 512 folders and files, the player allows access and navigates up to the maximum, but all items over the maximum are not accessible. Root Directory The root directory of the CD-R or CD-RW is treated as a folder. If the root directory has compressed audio files, the directory displays as the CD label. All files contained directly under the root directory are accessed prior to any root directory folders. However, playlists (Px) are always accessed before root folders or files. If a disc contains both uncompressed CD audio (.CDA) and MP3 files, a folder under the root directory called CD accesses all of the CD audio tracks on the disc. Empty Directory or Folder If a root directory or a folder exists somewhere in the file structure that contains only folders/subfolders and no compressed files directly beneath them, the player advances to the next folder in the file structure that contains compressed audio files. The empty folder does not display. No Folder When the CD contains only compressed files, the files are located under the root folder. The next and previous folder function does not display on a CD that was recorded without folders or playlists. When the CD contains only playlists and compressed audio files, but no folders, all files are located under the root folder. The folder down and up buttons search playlists (Px) first and then goes to the root folder. Order of Play Tracks recorded to the CD-R or CD-RW are played in the following order: · Play begins from the first track in the first playlist and continues sequentially through all tracks in each playlist. When the last track of the last playlist has played, play continues from the first track of the first playlist. · Play begins from the first track in the first folder and continues sequentially through all tracks in each folder. When the last track of the last folder has played, play continues from the first track of the first folder. When play enters a new folder, the display does not automatically show the new folder name unless the folder mode has been chosen as the default display. The new track name displays. 3-109 File System and Naming The song name that displays is the song name that is contained in the ID3 tag. If the song name is not present in the ID3 tag, then the radio displays the file name without the extension (such as .mp3) as the track name. Track names longer than 32 characters or four pages are shortened. Parts of words on the last page of text and the extension of the filename does not display. Preprogrammed Playlists Preprogrammed playlists that were created using WinAmpTM, MusicMatchTM, or Real JukeboxTM software can be accessed, however, they cannot be edited using the radio. These playlists are treated as special folders containing compressed audio song files. Playing an MP3 Insert a CD-R or CD-RW partway into the slot (Single CD Player), or press the load button and wait for the message to insert disc (Six-Disc CD Player), label side up. The player pulls it in, and the CD-R or CD-RW should begin playing. If the ignition or radio is turned off with a CD-R or CD-RW in the player it stays in the player. When the ignition or radio is turned on, the CD-R or CD-RW starts to play where it stopped, if it was the last selected audio source. As each new track starts to play, the track number and song title displays. 3-110 Z EJECT: Press this button to eject CD-R(s) or CD-RW(s). To eject the CD-R or CD-RW that is currently playing, press and release this button. A beep sounds and Ejecting Disc displays. Once the disc is ejected, Remove Disc displays. The CD-R or CD-RW can be removed. If the CD-R or CD-RW is not removed, after several seconds, the CD-R or CD-RW automatically pulls back into the player and begins playing. For the Six-Disc CD player, press and hold this button for two seconds to eject all discs. f (Tune): Turn this knob to select MP3 files on the CD-R currently playing. ©SEEK ¨: Press the left SEEK arrow to go to the start of the current MP3 file, if more than ten seconds have played. Press the right SEEK arrow to go to the next MP3 file. If either SEEK arrow is held or pressed multiple times, the player continues moving backward or forward through MP3 files on the CD. S c (Previous Folder): Press the pushbutton positioned under the Folder label to go to the first track in the previous folder. c T (Next Folder): Press the pushbutton positioned under the Folder label to go to the first track in the next folder. s REV (Reverse): Press and hold this button to reverse playback quickly within an MP3 file. Sound will be heard at a reduced volume. Release this button to resume playing the file. The elapsed time of the file displays. \ FWD (Fast Forward): Press and hold this button to advance playback quickly within an MP3 file. Sound will be heard at a reduced volume. Release this button to resume playing the file. The elapsed time of the file displays. RDM (Random): With the random setting, MP3 files on the CD-R or CD-RW can be listened to in random, rather than sequential order, on one CD-R or CD-RW or all discs in a six-disc CD player. To use random, do one of the following: 1. To play MP3 files from the CD-R or CD-RW in random order, press the pushbutton positioned under the RDM label until Random Current Disc displays. Press the same pushbutton again to turn off random play. 2. To play songs from all CDs loaded in a six-disc CD player in random order, press the pushbutton positioned under the RDM label until Randomize All Discs displays. Press the same pushbutton again to turn off random play. h (Music Navigator): Use the music navigator feature to play MP3 files on the CD-R or CD-RW in order by artist or album. Press the pushbutton located below the music navigator label. The player scans the disc to sort the files by artist and album ID3 tag information. It could take several minutes to scan the disc depending on the number of MP3 files recorded to the CD-R or CD-RW. The radio can begin playing while it is scanning the disc in the background. When the scan is finished, the CD-R or CD-RW begins playing again. Once the disc has scanned, the player defaults to playing MP3 files in order by artist. The current artist playing is shown on the second line of the display between the arrows. Once all songs by that artist are played, the player moves to the next artist in alphabetical order on the CD-R or CD-RW and begins playing MP3 files by that artist. To listen to MP3 files by another artist, press the pushbutton located below either arrow button. The next or previous artist in alphabetical order plays. Continue pressing either button until the desired artist is displayed. 3-111 To change from playback by artist to playback by album, press the pushbutton located below the Sort By label. From the sort screen, push one of the buttons below the album button. Press the pushbutton below the back label to return to the main music navigator screen. Now the album name is displayed on the second line between the arrows and songs from the current album begins to play. Once all songs from that album are played, the player moves to the next album in alphabetical order on the CD-R or CD-RW and begins playing MP3 files from that album. To exit music navigator mode, press the pushbutton below the Back label to return to normal MP3 playback. BAND: Press this button to listen to the radio when a CD is playing. The CD remains safely inside the radio for future listening. CD/AUX (CD/Auxiliary): Press this button to play a CD when listening to the radio. The CD icon and a message showing disc and/or track number displays when a CD is in the player. Press this button again and the system automatically searches for an auxiliary input device such as a portable audio player. If a portable audio player is not connected, "No Input Device Found" displays. Using an MP3 (Radio with CD and DVD Player) MP3/WMA CD-R or CD-RW Disc Compressed Audio or Mixed Mode Discs The radio also plays discs that contain both uncompressed CD audio (.CDA files) and MP3/WMA files depending on which slot the disc is loaded into. By default the radio reads only the uncompressed audio (.CDA) and ignores the MP3/WMA files on the DVD deck. On the CD deck, press the CAT (category) button to toggle between compressed and uncompressed audio format, the default being the uncompressed format (.CDA). MP3/WMA Format Burning an MP3/WMA disc on a personal computer: · Make sure the MP3/WMA files are recorded on a CD-R or CD-RW disc. · Do not mix standard audio and MP3/WMA files on one disc. · The CD player (lower slot) is able to read and play a maximum combination of 512 files and folders. The DVD player (upper slot) is able to read 255 folders, 15 playlists and 40 sessions. 3-112 · Create a folder structure that makes it easy to find songs while driving. Organize songs by albums using one folder for each album. Each folder or album should contain 18 songs or less. · Avoid subfolders. The system can support up to eight subfolders deep, however, keep the total number of folders to a minimum in order to reduce the complexity and confusion in trying to locate a particular folder during playback. · Make sure playlists have a .m3u extension as other file extensions might not work. · Minimize the length of the file, folder, or playlist names. Long file, folder, or playlist names, or a combination of a large number of files and folders, or playlists could cause the player to be unable to play up to the maximum number of files, folders, playlists, or sessions. To play a large number of files, folders, playlists, or sessions, minimize the length of the file, folder, or playlist name. Long names also take up more space on the display, potentially getting cut off. · Finalize the audio disc before burning it. Trying to add music to an existing disc could cause the disc not to function in the player. Root Directory The root directory of the CD-R or CD-RW is treated as a folder. If the root directory has compressed audio files, the directory is displayed as F1 ROOT. All files contained directly under the root directory are accessed prior to any root directory folders. However, playlists (Px) are always accessed before root folders or files. Empty Directory or Folder If a root directory or a folder exists somewhere in the file structure that contains only folders/subfolders and no compressed files directly beneath them, the player advances to the next folder in the file structure that contains compressed audio files. The empty folder does not display. No Folder When the CD contains only compressed files, the files are located under the root folder. The next and previous folder function does not function on a CD that was recorded without folders or playlists. When displaying the name of the folder the radio displays ROOT. When the CD contains only playlists and compressed audio files, but no folders, all files are located under the root folder. The folder down and the folder up buttons search playlists (Px) first and then goes to the root folder. When the radio displays the name of the folder the radio displays ROOT. 3-113 Order of Play Tracks recorded to the CD-R or CD-RW are played in the following order: · Play begins from the first track in the first playlist and continues sequentially through all tracks in each playlist. When the last track of the last playlist has played, play continues from the first track of the first playlist. · Play begins from the first track in the first folder and continues sequentially through all tracks in each folder. When the last track of the last folder has played, play continues from the first track of the first folder. When play enters a new folder, the display does not automatically show the new folder name unless folder mode has been chosen as the default display. The new track name displays. File System and Naming The song name that is displayed is the song name that is contained in the ID3 tag. If the song name is not present in the ID3 tag, then the radio displays the file name without the extension (such as .mp3) as the track name. Track names longer than 32 characters or four pages are shortened. Parts of words on the last page of text and the extension of the filename displays. Preprogrammed Playlists Preprogrammed playlists that were created using WinAmpTM, MusicMatchTM, or Real JukeboxTM software can be accessed, however, they cannot be edited using the radio. These playlists are treated as special folders containing compressed audio song files. Playing an MP3/WMA (In Either the DVD or CD Slot) Insert a CD-R or CD-RW partway into either the top or bottom slot, ify the reading. 5. If the fluid level is below the COLD check band, add only enough fluid as necessary to bring the level into the COLD band. It does not take much fluid, generally less than one pint (0.5L). Do not overfill. 6. Perform a hot check at the first opportunity after the transmission reaches a normal operating temperature between 160°F to 200°F (71°C to 93°C). 7. If the fluid level is in the acceptable range, push the dipstick back in all the way, then flip the handle down to lock the dipstick in place. 5-26 Hot Check Procedure Use this procedure to check the transmission fluid level when the transmission fluid temperature is between 160°F and 200°F (71°C and 93°C). The hot check is the most accurate method to check the fluid level. The hot check should be performed at the first opportunity in order to verify the cold check. The fluid level rises as fluid temperature increases, so it is important to ensure the transmission temperature is within range. 1. Locate the transmission dipstick at the rear of the engine compartment, on the passenger side of the vehicle. See Engine Compartment Overview on page 5-14 for more information. 2. Flip the handle up, and then pull out the dipstick and wipe it with a clean rag or paper towel. 3. Install the dipstick by pushing it back in all the way, wait three seconds, and then pull it back out again. 4. Check both sides of the dipstick and read the lower level. Repeat the check procedure to verify the reading. 5. Safe operating level is within the HOT cross hatch band on the dipstick. If the fluid level is not within the HOT band, and the transmission temperature is between 160°F and 200°F (71°C and 93°C), add or drain fluid as necessary to bring the level into the HOT band. If the fluid level is low, add only enough fluid to bring the level into the HOT band. It does not take much fluid, generally less than one pint (0.5L). Do not overfill. 6. If the fluid level is in the acceptable range, push the dipstick back in all the way, then flip the handle down to lock the dipstick in place. 5-27 Consistency of Readings Always check the fluid level at least twice using the procedure described previously. Consistency (repeatable readings) is important to maintaining proper fluid level. If readings are still inconsistent, contact your dealer/retailer. Cooling System If your vehicle has the DURAMAX® Diesel engine, see the DURAMAX® Diesel manual for more information. The Cooling System allows the engine to maintain the correct working temperature. 5.3L Engine (4.3L, 4.8L, 6.0L and 6.2L Similar) A. Coolant Surge Tank B. Coolant Surge Tank Pressure Cap C. Engine Cooling Fan 5-28 { CAUTION: An electric engine cooling fan can start even when the engine is not running. To avoid injury, always keep hands, clothing, and tools away from any engine cooling fan. { CAUTION: Heater and radiator hoses, and other engine parts, can be very hot. Do not touch them. If you do, you can be burned. Do not run the engine if there is a leak. If you run the engine, it could lose all coolant. That could cause an engine fire, and you could be burned. Get any leak fixed before you drive the vehicle. Notice: Using coolant other than DEX-COOL® can cause premature engine, heater core, or radiator corrosion. In addition, the engine coolant could require changing sooner, at 50 000 km (30,000 miles) or 24 months, whichever occurs first. Any repairs would not be covered by the vehicle warranty. Always use DEX-COOL® (silicate-free) coolant in the vehicle. Engine Coolant The cooling system in the vehicle is filled with DEX-COOL® engine coolant. This coolant is designed to remain in the vehicle for five years or 150,000 miles (240 000 km), whichever occurs first. The following explains the cooling system and how to check and add coolant when it is low. If there is a problem with engine overheating, see Engine Overheating on page 5-34. 5-29 What to Use { CAUTION: Adding only plain water to the cooling system can be dangerous. Plain water, or some other liquid such as alcohol, can boil before the proper coolant mixture will. The vehicle's coolant warning system is set for the proper coolant mixture. With plain water or the wrong mixture, the engine could get too hot but you would not get the overheat warning. The engine could catch fire and you or others could be burned. Use a 50/50 mixture of clean, drinkable water and DEX-COOL® coolant. Use a 50/50 mixture of clean, drinkable water and DEX-COOL® coolant. If using this mixture, nothing else needs to be added. This mixture: · Gives freezing protection down to -34°F (-37°C), outside temperature. · Gives boiling protection up to 265°F (129°C), engine temperature. · Protects against rust and corrosion. · Will not damage aluminum parts. · Helps keep the proper engine temperature. Notice: If an improper coolant mixture is used, the engine could overheat and be badly damaged. The repair cost would not be covered by the vehicle warranty. Too much water in the mixture can freeze and crack the engine, radiator, heater core, and other parts. Notice: If extra inhibitors and/or additives are used in the vehicle's cooling system, the vehicle could be damaged. Use only the proper mixture of the engine coolant listed in this manual for the cooling system. See Recommended Fluids and Lubricants on page 6-15 for more information. 5-30 Checking Coolant The coolant surge tank is located in the engine compartment on the passenger side of the vehicle. See Engine Compartment Overview on page 5-14 for more information on location. The vehicle must be on a level surface when checking the coolant level. Check to see if coolant is visible in the coolant surge tank. If the coolant inside the coolant surge tank is boiling, do not do anything else until it cools down. If coolant is visible but the coolant level is not at or above the FULL COLD mark, add a 50/50 mixture of clean, drinkable water and DEX-COOL® coolant at the coolant surge tank, but be sure the cooling system is cool before this is done. The coolant level should be at or above the FULL COLD mark. If it is not, you may have a leak in the cooling system. If the vehicle has a low coolant sensor and the LOW COOLANT LEVEL message comes on and stays on, it means you are low on engine coolant. See "LOW COOLANT LEVEL" under DIC Warnings and Messages on page 3-66. 5-31 How to Add Coolant to the Coolant Surge Tank for Gasoline Engines If the vehicle has a diesel engine, see "Cooling System" in the DURAMAX® Diesel Supplement for the proper coolant fill procedure. { CAUTION: You can be burned if you spill coolant on hot engine parts. Coolant contains ethylene glycol and it will burn if the engine parts are hot enough. Do not spill coolant on a hot engine. Notice: This vehicle has a specific coolant fill procedure. Failure to follow this procedure could cause the engine to overheat and be severely damaged. { CAUTION: An electric engine cooling fan under the hood can start up even when the engine is not running and can cause injury. Keep hands, clothing, and tools away from any underhood electric fan. 5-32 { CAUTION: Steam and scalding liquids from a hot cooling system can blow out and burn you badly. They are under pressure, and if you turn the coolant surge tank pressure cap -- even a little -- they can come out at high speed. Never turn the cap when the cooling system, including the coolant surge tank pressure cap, is hot. Wait for the cooling system and coolant surge tank pressure cap to cool if you ever have to turn the pressure cap. If no coolant is visible in the surge tank, add coolant as follows: 1. Remove the coolant surge tank pressure cap when the cooling system, including the coolant surge tank pressure cap and upper radiator hose, is no longer hot. Turn the pressure cap slowly counterclockwise about one full turn. If you hear a hiss, wait for that to stop. A hiss means there is still some pressure left. 2. Keep turning the pressure cap slowly, and remove it. 5-33 3. Fill the coolant surge tank with the proper mixture to the FULL COLD mark. 4. With the coolant surge tank pressure cap off, start the engine and let it run until the engine coolant temperature gage indicates approximately 195°F (90°C). By this time, the coolant level inside the coolant surge tank may be lower. If the level is lower, add more of the proper mixture to the coolant surge tank until the level reaches the FULL COLD mark. 5. Replace the pressure cap. Be sure the pressure cap is hand-tight and fully seated. 6. Verify coolant level after engine is shut off and the coolant is cold. If necessary, repeat coolant fill procedure Steps 1 through 6. Notice: If the pressure cap is not tightly installed, coolant loss and possible engine damage may occur. Be sure the cap is properly and tightly secured. Engine Overheating If the vehicle has the DURAMAX® Diesel engine, see the DURAMAX® Diesel manual for more information. The vehicle has several indicators to warn of engine overheating. You will find a coolant temperature gage on the vehicle's instrument panel. See Engine Coolant Temperature Gage (US-Canada) on page 3-44. In addition, you will find ENGINE OVERHEATED STOP ENGINE, ENGINE OVERHEATED IDLE ENGINE, and ENGINE POWER IS REDUCED messages in the Driver Information Center (DIC) on the instrument panel. See DIC Warnings and Messages on page 3-66. 5-34 You may decide not to lift the hood when this warning appears, but instead get service help right away. See Roadside Assistance Program on page 7-7. If you do decide to lift the hood, make sure the vehicle is parked on a level surface. Then check to see if the engine cooling fans are running. If the engine is overheating, both fans should be running. If they are not, do not continue to run the engine and have the vehicle serviced. Notice: Engine damage from running your engine without coolant is not covered by your warranty. See Overheated Engine Protection Operating Mode on page 5-37 for information on driving to a safe place in an emergency. Notice: If the engine catches fire while driving with no coolant, the vehicle can be badly damaged. The costly repairs would not be covered by the vehicle warranty. See Overheated Engine Protection Operating Mode on page 5-37 for information on driving to a safe place in an emergency. If Steam Is Coming From The Engine Compartment { CAUTION: Steam from an overheated engine can burn you badly, even if you just open the hood. Stay away from the engine if you see or hear steam coming from it. Turn it off and get everyone away from the vehicle until it cools down. Wait until there is no sign of steam or coolant before you open the hood. If you keep driving when the vehicles engine is overheated, the liquids in it can catch fire. You or others could be badly burned. Stop the engine if it overheats, and get out of the vehicle until the engine is cool. See Overheated Engine Protection Operating Mode on page 5-37 for information on driving to a safe place in an emergency. 5-35 If No Steam Is Coming From The Engine Compartment The ENGINE OVERHEATED STOP ENGINE or the ENGINE OVERHEATED IDLE ENGINE message, along with a low coolant condition, can indicate a serious problem. If you get an engine overheat warning, but see or hear no steam, the problem may not be too serious. Sometimes the engine can get a little too hot when you: · Climb a long hill on a hot day. · Stop after high-speed driving. · Idle for long periods in traffic. · Tow a trailer. See Towing a Trailer on page 4-50. If you get the ENGINE OVERHEATED STOP ENGINE or the ENGINE OVERHEATED IDLE ENGINE message with no sign of steam, try this for a minute or so: 1. Turn the air conditioning off. 2. Turn the heater on to the highest temperature and to the highest fan speed. Open the windows as necessary. 3. If you are stopped in a traffic jam, apply the brake, shift to N (Neutral); otherwise, shift to the highest gear while driving -- D (Drive) or 3 (Third). If the temperature overheat gage is no longer in the overheat zone or an overheat warning no longer displays, the vehicle can be driven. Continue to drive the vehicle slow for about 10 minutes. Keep a safe vehicle distance from the car in front of you. If the warning does not come back on, continue to drive normally. If the warning continues, pull over, stop, and park the vehicle right away. If there is no sign of steam, idle the engine for five minutes while parked. If the warning is still displayed, turn off the engine until it cools down. Also, see "Overheated Engine Protection Operating Mode" later in this section. 5-36 Overheated Engine Protection Operating Mode If an overheated engine condition exists and the REDUCED ENGINE POWER message is displayed, an overheat protection mode which alternates firing groups of cylinders helps prevent engine damage. In this mode, you will notice a loss in power and engine performance. This operating mode allows the vehicle to be driven to a safe place in an emergency. Driving extended miles (km) and/or towing a trailer in the overheat protection mode should be avoided. Notice: After driving in the overheated engine protection operating mode, to avoid engine damage, allow the engine to cool before attempting any repair. The engine oil will be severely degraded. Repair the cause of coolant loss, change the oil and reset the oil life system. See Engine Oil on page 5-15. Engine Fan Noise If the vehicle has a clutched engine cooling fan, when the clutch is engaged, the fan spins faster to provide more air to cool the engine. In most everyday driving conditions, the fan is spinning slower and the clutch is not fully engaged. This improves fuel economy and reduces fan noise. Under heavy vehicle loading, trailer towing, and/or high outside temperatures, the fan speed increases as the clutch more fully engages, so you may hear an increase in fan noise. This is normal and should not be mistaken as the transmission slipping or making extra shifts. It is merely the cooling system functioning properly. The fan will slow down when additional cooling is not required and the clutch disengages. You may also hear this fan noise when you start the engine. It will go away as the fan clutch partially disengages. If the vehicle has electric cooling fans, you may hear the fans spinning at low speed during most everyday driving. The fans may turn off if no cooling is required. Under heavy vehicle loading, trailer towing, and/or high outside temperatures, or if you are operating the air conditioning system, the fans may change to high speed and you may hear an increase in fan noise. This is normal and indicates that the cooling system is functioning properly. The fans will change to low speed when additional cooling is no longer required. 5-37 Power Steering Fluid See Engine Compartment Overview on page 5-14 for reservoir location. When to Check Power Steering Fluid It is not necessary to regularly check power steering fluid unless you suspect there is a leak in the system or you hear an unusual noise. A fluid loss in this system could indicate a problem. Have the system inspected and repaired. How to Check Power Steering Fluid To check the power steering fluid: 1. Turn the key off and let the engine compartment cool down. 2. Wipe the cap and the top of the reservoir clean. 3. Unscrew the cap and wipe the dipstick with a clean rag. 4. Replace the cap and completely tighten it. 5. Remove the cap again and look at the fluid level on the dipstick. The level should be at the FULL COLD mark. If necessary, add only enough fluid to bring the level up to the mark. What to Use To determine what kind of fluid to use, see Recommended Fluids and Lubricants on page 6-15. Always use the proper fluid. Notice: Use of the incorrect fluid may damage the vehicle and the damages may not be covered by the vehicle's warranty. Always use the correct fluid listed in Recommended Fluids and Lubricants on page 6-15. 5-38 Windshield Washer Fluid What to Use When windshield washer fluid needs to be added, be sure to read the manufacturer's instructions before use. Use a fluid that has sufficient protection against freezing in an area where the temperature may fall below freezing. Adding Washer Fluid The vehicle has a low washer fluid message in the DIC that comes on when the washer fluid is low. The message is displayed for 15 seconds at the start of each ignition cycle. When the WASHER FLUID LOW ADD FLUID message displays, you will need to add washer fluid to the windshield washer fluid reservoir. Open the cap with the washer symbol on it. Add washer fluid until the tank is full. See Engine Compartment Overview on page 5-14 for reservoir location. Notice: · When using concentrated washer fluid, follow the manufacturer's instructions for adding water. · Do not mix water with ready-to-use washer fluid. Water can cause the solution to freeze and damage your washer fluid tank and other parts of the washer system. Also, water does not clean as well as washer fluid. · Fill the washer fluid tank only three-quarters full when it is very cold. This allows for fluid expansion if freezing occurs, which could damage the tank if it is completely full. · Do not use engine coolant (antifreeze) in your windshield washer. It can damage the vehicle's windshield washer system and paint. 5-39 Brakes Brake Fluid The brake master cylinder reservoir is filled with DOT 3 brake fluid. See Engine Compartment Overview on page 5-14 for the location of the reservoir. There are only two reasons why the brake fluid level in the reservoir might go down: · The brake fluid level goes down because of normal brake lining wear. When new linings are installed, the fluid level goes back up. · A fluid leak in the brake hydraulic system can also cause a low fluid level. Have the brake hydraulic system fixed, since a leak means that sooner or later the brakes will not work well. Do not top off the brake fluid. Adding fluid does not correct a leak. If fluid is added when the linings are worn, there will be too much fluid when new brake linings are installed. Add or remove brake fluid, as necessary, only when work is done on the brake hydraulic system. { CAUTION: If too much brake fluid is added, it can spill on the engine and burn, if the engine is hot enough. You or others could be burned, and the vehicle could be damaged. Add brake fluid only when work is done on the brake hydraulic system. See "Checking Brake Fluid" in this section. Refer to the Maintenance Schedule to determine when to check the brake fluid. See Scheduled Maintenance (Gasoline Engine) on page 6-4. 5-40 Checking Brake Fluid Check brake fluid by looking at the brake fluid reservoir. See Engine Compartment Overview on page 5-14. The fluid level should be above MIN. If it is not, have the brake hydraulic system checked to see if there is a leak. After work is done on the brake hydraulic system, make sure the level is above the MIN but not over the MAX mark. What to Add Use only new DOT 3 brake fluid from a sealed container. See Recommended Fluids and Lubricants on page 6-15. Always clean the brake fluid reservoir cap and the area around the cap before removing it. This helps keep dirt from entering the reservoir. { CAUTION: With the wrong kind of fluid in the brake hydraulic system, the brakes might not work well. This could cause a crash. Always use the proper brake fluid. Notice: · Using the wrong fluid can badly damage brake hydraulic system parts. For example, just a few drops of mineral-based oil, such as engine oil, in the brake hydraulic system can damage brake hydraulic system parts so badly that they will have to be replaced. Do not let someone put in the wrong kind of fluid. · If brake fluid is spilled on the vehicle's painted surfaces, the paint finish can be damaged. Be careful not to spill brake fluid on the vehicle. If you do, wash it off immediately. See Washing Your Vehicle on page 5-116. 5-41 Brake Wear This vehicle has front disc brakes and could have rear drum brakes or rear disc brakes. Disc brake pads have built-in wear indicators that make a high-pitched warning sound when the brake pads are worn and new pads are needed. The sound can come and go or be heard all the time the vehicle is moving, except when applying the brake pedal firmly. { CAUTION: The brake wear warning sound means that soon the brakes will not work well. That could lead to an accident. When the brake wear warning sound is heard, have the vehicle serviced. Notice: Continuing to drive with worn-out brake pads could result in costly brake repair. Some driving conditions or climates can cause a brake squeal when the brakes are first applied or lightly applied. This does not mean something is wrong with the brakes. Properly torqued wheel nuts are necessary to help prevent brake pulsation. When tires are rotated, inspect brake pads for wear and evenly tighten wheel nuts in the proper sequence to torque specifications in Capacities and Specifications on page 5-129. If the vehicle has rear drum brakes, they do not have wear indicators, but if a rear brake rubbing noise is heard, have the rear brake linings inspected immediately. Rear brake drums should be removed and inspected each time the tires are removed for rotation or changing. Drum brakes have an inspection hole to inspect lining wear during scheduled maintenance. When the front brake pads are replaced, have the rear brakes inspected, too. Brake linings should always be replaced as complete axle sets. Brake Pedal Travel See your dealer/retailer if the brake pedal does not return to normal height, or if there is a rapid increase in pedal travel. This could be a sign that brake service might be required. Brake Adjustment Every brake stop, the brakes adjust for wear. 5-42 Replacing Brake System Parts The braking system on a vehicle is complex. Its many parts have to be of top quality and work well together if the vehicle is to have really good braking. The vehicle was designed and tested with top-quality brake parts. When parts of the braking system are replaced -- for example, when the brake linings wear down and new ones are installed -- be sure to get new approved replacement parts. If this is not done, the brakes might not work properly. For example, if someone puts in brake linings that are wrong for the vehicle, the balance between the front and rear brakes can change -- for the worse. The braking performance expected can change in many other ways if the wrong replacement brake parts are installed. Battery If the vehicle is a Two-mode Hybrid, see the Two-mode Hybrid manual for more information. This vehicle has a maintenance free battery (or batteries). When it is time for a new battery, see your dealer/retailer for one that has the replacement number shown on the original battery's label. See Engine Compartment Overview on page 5-14 for battery location. Warning: Battery posts, terminals, and related accessories contain lead and lead compounds, chemicals known to the State of California to cause cancer and reproductive harm. Wash hands after handling. Vehicle Storage { CAUTION: Batteries have acid that can burn you and gas that can explode. You can be badly hurt if you are not careful. See Jump Starting on page 5-44 for tips on working around a battery without getting hurt. Infrequent Usage: If the vehicle is driven infrequently, remove the black, negative (-) cable from the battery. This helps keep the battery from running down. Extended Storage: For extended storage of the vehicle, remove the black, negative (-) cable from the battery or use a battery trickle charger. This helps maintain the charge of the battery over an extended period of time. 5-43 Jump Starting If the vehicle is a Two-mode Hybrid, see the Two-mode Hybrid manual for more information. If the vehicle's battery (or batteries) has run down, you may want to use another vehicle and some jumper cables to start your vehicle. Be sure to use the following steps to do it safely. { CAUTION: Batteries can hurt you. They can be dangerous because: · They contain acid that can burn you. · They contain gas that can explode or ignite. · They contain enough electricity to burn you. If you do not follow these steps exactly, some or all of these things can hurt you. Notice: Ignoring these steps could result in costly damage to the vehicle that would not be covered by the warranty. Trying to start the vehicle by pushing or pulling it will not work, and it could damage the vehicle. 1. Check the other vehicle. It must have a 12-volt battery with a negative ground system. Notice: If the other vehicle's system is not a 12-volt system with a negative ground, both vehicles can be damaged. Only use vehicles with 12-volt systems with negative grounds to jump start your vehicle. 2. If you have a vehicle with a diesel engine with two batteries, you should know before you begin that, especially in cold weather, you may not be able to get enough power from a single battery in another vehicle to start your diesel engine. If your vehicle has more than one battery, use the battery that is closer to the starter -- this will reduce electrical resistance. This is located on the passenger side, in the rear of the engine compartment. 5-44 3. Get the vehicles close enough so the jumper cables can reach, but be sure the vehicles are not touching each other. If they are, it could cause an unwanted ground connection. You would not be able to start your vehicle, and the bad grounding could damage the electrical systems. To avoid the possibility of the vehicles rolling, set the parking brake firmly on both vehicles involved in the jump start procedure. Put the automatic transmission in P (Park) or a manual transmission in Neutral before setting the parking brake. If you have a four-wheel-drive vehicle, be sure the transfer case is in a drive gear, not in Neutral. Notice: If you leave the radio or other accessories on during the jump starting procedure, they could be damaged. The repairs would not be covered by the warranty. Always turn off the radio and other accessories when jump starting the vehicle. 4. Turn off the ignition on both vehicles. Unplug unnecessary accessories plugged into the cigarette lighter or the accessory power outlets. Turn off the radio and all the lamps that are not needed. This will avoid sparks and help save both batteries. And it could save the radio! 5. Open the hood on the other vehicle and locate the positive (+) and negative (-) terminal locations on that vehicle. The positive (+) terminal, is located under a red plastic cover at the positive battery post. To uncover the positive (+) terminal, open the red plastic cover. 5-45 The remote negative (-) terminal is a stud located on the right front passenger side of the engine, where the negative battery cable attaches. See Engine Compartment Overview on page 5-14. { CAUTION: An electric fan can start up even when the engine is not running and can injure you. Keep hands, clothing and tools away from any underhood electric fan. 5-46 { CAUTION: Using a match near a battery can cause battery gas to explode. People have been hurt doing this, and some have been blinded. Use a flashlight if you need more light. Be sure the battery has enough water. You do not need to add water to the battery installed in your new vehicle. But if a battery has filler caps, be sure the right amount of fluid is there. If it is low, add water to take care of that first. If you do not, explosive gas could be present. Battery fluid contains acid that can burn you. Do not get it on you. If you accidentally get it in your eyes or on your skin, flush the place with water and get medical help immediately. { CAUTION: Fans or other moving engine parts can injure you badly. Keep your hands away from moving parts once the engine is running. 7. Connect the red positive (+) cable to the positive (+) terminal of the vehicle with the dead battery. 6. Check that the jumper cables do not have loose or missing insulation. If they do, you could get a shock. The vehicles could be damaged too. Before you connect the cables, here are some basic things you should know. Positive (+) will go to positive (+) or to a remote positive (+) terminal if the vehicle has one. Negative (-) will go to a heavy, unpainted metal engine part or to a remote negative (-) terminal if the vehicle has one. Do not connect positive (+) to negative (-) or you will get a short that would damage the battery and maybe other parts too. And do not connect the negative (-) cable to the negative (-) terminal on the dead battery because this can cause sparks. 5.3L engine (4.3L, 4.8L, 6.0L and 6.2L similar) 8. Do not let the other end touch metal. Connect it to the positive (+) terminal of the good battery. Use a remote positive (+) terminal if the vehicle has one. 9. Now connect the black negative (-) cable to the negative (-) terminal of the good battery. Use a remote negative (-) terminal if the vehicle has one. Do not let the other end touch anything until the next step. 5-47 10. Connect the other end of the negative (-) cable to a heavy, unpainted metal engine part or to the remote negative (-) terminal, on the vehicle with the dead battery. 11. Start the vehicle with the good battery and run the engine for a while. 12. Try to start the vehicle that had the dead battery. If it will not start after a few tries, it probably needs service. Notice: If the jumper cables are connected or removed in the wrong order, electrical shorting may occur and damage the vehicle. The repairs would not be covered by the vehicle warranty. Always connect and remove the jumper cables in the correct order, making sure that the cables do not touch each other or other metal. Jumper Cable Removal A. Heavy, Unpainted Metal Engine Part or Remote Negative (-) Terminal B. Good Battery or Remote Positive (+) and Remote Negative (-) Terminals C. Dead Battery or Remote Positive (+) Terminal 5-48 To disconnect the jumper cables from both vehicles do the following: 1. Disconnect the black negative (-) cable from the vehicle that had the bad battery. 2. Disconnect the black negative (-) cable from the vehicle with the good battery. 3. Disconnect the red positive (+) cable from the vehicle with the good battery. 4. Disconnect the red positive (+) cable from the other vehicle. 5. Return the positive (+) terminal cover, to its original position. Rear Axle When to Check Lubricant It is not necessary to regularly check rear axle fluid unless you suspect there is a leak or you hear an unusual noise. A fluid loss could indicate a problem. Have it inspected and repaired. All axle assemblies are filled by volume of fluid during production. They are not filled to reach a certain level. When checking the fluid level on any axle, variations in the readings can be caused by factory fill differences between the minimum and the maximum fluid volume. Also, if a vehicle has just been driven before checking the fluid level, it may appear lower than normal because fluid has traveled out along the axle tubes and has not drained back to the sump area. Therefore, a reading taken five minutes after the vehicle has been driven will appear to have a lower fluid level than a vehicle that has been stationary for an hour or two. Remember that the rear axle assembly must be supported to get a true reading. How to Check Lubricant 2500HD with 6.0L and 6.2L 5-49 All Other Series and Engines To get an accurate reading, the vehicle should be on a level surface. · For all 4.3L, 4.8L and 5.3L 1500 Series applications, the proper level is 0.04 inches to 0.75 inches (1.0 mm to 19.0 mm) below the bottom of the filler hole, located on the rear axle. Add only enough fluid to reach the proper level. · For all 6.0L and 6.2L 1500 Series applications, the proper level is from 0.6 inches to 1.6 inches (15 mm to 40 mm) below the bottom of the filler plug hole, located on the rear axle. Add only enough fluid to reach the proper level. 5-50 · For all 6.0L and 6.2L 2500HD Series applications, the proper level is from 0 to 0.5 inches (0 mm to 13 mm) below the bottom of the filler plug hole, located on the rear axle. Add only enough fluid to reach the proper level. · For all 6.6L DURAMAX DIESEL 2500HD Series applications and all 3500 Series applications, the proper level is from 0.6 inches to 0.8 inches (17 mm to 21 mm) below the bottom of the filler plug hole, located on the rear axle. Add only enough fluid to reach the proper level. What to Use Refer to the Maintenance Schedule to determine what kind of lubricant to use. See Recommended Fluids and Lubricants on page 6-15. Four-Wheel Drive Lubricant checks in this section also apply to these vehicles. Transfer Case When to Check Lubricant It is not necessary to regularly check transfer case fluid unless you suspect there is a leak, or you hear an unusual noise. A fluid loss could indicate a problem. Have it inspected and repaired. How to Check Lubricant Electric Shift Transfer Case A. Fill Plug B. Drain Plug Manual Shift Transfer Case A. Fill Plug B. Drain Plug 5-51 Active Transfer Case A. Fill Plug B. Drain Plug To get an accurate reading, the vehicle should be on a level surface. If the level is below the bottom of the filler plug hole, located on the transfer case, you will need to add some lubricant. Add enough lubricant to raise the level to the bottom of the filler plug hole. Use care not to overtighten the plug. When to Change Lubricant Refer to the Maintenance Schedule to determine how often to change the lubricant. See Scheduled Maintenance (Gasoline Engine) on page 6-4. What to Use Refer to the Maintenance Schedule to determine what kind of lubricant to use. See Recommended Fluids and Lubricants on page 6-15. Front Axle When to Check and Change Lubricant It is not necessary to regularly check front axle fluid unless you suspect there is a leak or you hear an unusual noise. A fluid loss could indicate a problem. Have it inspected and repaired. How to Check Lubricant To get an accurate reading, the vehicle should be on a level surface. 5-52 A: Fill Plug B: Drain Plug 1500 Series All except 1500 Series A: Fill Plug B: Drain Plug · When the differential is cold, add enough lubricant to raise the level from 0 (0 mm) to 1/8 inch (3.2 mm) below the filler plug hole. · When the differential is at operating temperature (warm), add enough lubricant to raise the level to the bottom of the filler plug hole. 5-53 What to Use Refer to the Maintenance Schedule to determine what kind of lubricant to use. See Recommended Fluids and Lubricants on page 6-15. Noise Control System Tampering with Noise Control System Prohibited The following information relates to compliance with federal noise emission standards for vehicles with a Gross Vehicle Weight Rating (GVWR) of more than 10,000 lbs (4 536 kg). The Maintenance Schedule provides information on maintaining the noise control system to minimize degradation of the noise emission control system during the life of your vehicle. The noise control system warranty is given in your warranty booklet. These standards apply only to vehicles sold in the United States. Federal law prohibits the following acts or the causing thereof: 1. The removal or rendering inoperative by any person, other than for purposes of maintenance, repair or replacement, of any device or element of design incorporated into any new vehicle for the purpose of noise control, prior to its sale or delivery to the ultimate purchaser or while it is in use; or 2. The use of the vehicle after such device or element of design has been removed or rendered inoperative by any person. Among those acts presumed to constitute tampering are the acts listed below. Insulation: · Removal of the noise shields or any underhood insulation. Engine: · Removal or rendering engine speed governor, if the vehicle has one, inoperative so as to allow engine speed to exceed manufacturer specifications. 5-54 Fan and Drive: · Removal of fan clutch, if the vehicle has one, or rendering clutch inoperative. · Removal of the fan shroud, if the vehicle has one. Air Intake: · Removal of the air cleaner silencer. · Modification of the air cleaner. Exhaust: · Removal of the muffler and/or resonator. · Removal of the exhaust pipes and exhaust pipe clamps. Headlamp Aiming The vehicle has a visual optical headlamp aiming system. The aim of the headlamps have been preset at the factory and should need no further adjustment. However, if the vehicle is damaged in a crash, the aim of the headlamps can be affected and adjustment can be necessary. If oncoming vehicles flash their high beams at you, this can mean the vertical aim of your headlamps needs to be adjusted. It is recommended that the vehicle is taken to your dealer/retailer for service if the headlamps need to be adjusted. It is possible however, to re-aim the headlamps as described. The vehicle should: · Be placed so the headlamps are 25 ft. (7.6 m) from a light colored wall. · Have all four tires on a level surface which is level all the way to the wall. · Be placed so it is perpendicular to the wall. · Not have any snow, ice, or mud on it. · Be fully assembled and all other work stopped while headlamp aiming is being performed. · Be normally loaded with a full tank of fuel and one person or 160 lbs (75 kg) sitting on the driver seat. · Have tires properly inflated. · Have the spare tire is in its proper location in the vehicle. 5-55 Headlamp aiming is done with the vehicle's low-beam headlamps. The high-beam headlamps will be correctly aimed if the low-beam headlamps are aimed properly. To adjust the vertical aim: 1. Open the hood. See Hood Release on page 5-13 for more information. 2. Locate the aim dot on the lens of the low-beam headlamp. 3. Record the distance from the ground to the aim dot on the low-beam headlamp. 4. At a wall measure from the ground upward (A) to the recorded distance from Step 3 and mark it. 5. Draw or tape a horizontal line (B) on the wall or flat surface the width of the vehicle at the height of the mark in Step 4. Notice: Do not cover a headlamp to improve beam cut-off when aiming. Covering a headlamp may cause excessive heat build-up which may cause damage to the headlamp. 6. Turn on the low-beam headlamps and place a piece of cardboard or equivalent in front of the headlamp not being adjusted. This allows only the beam of light from the headlamp being adjusted to be seen on the flat surface. 5-56 7. Locate the vertical headlamp aiming screws, which are under the hood near each headlamp assembly. The adjustment screw can be turned with a E8 Torx® socket. 8. Turn the vertical aiming screw until the headlamp beam is aimed to the horizontal tape line. Turn it clockwise or counterclockwise to raise or lower the angle of the beam. 9. Make sure that the light from the headlamp is positioned at the bottom edge of the horizontal tape line. The lamp on the left (A) shows the correct headlamp aim. The lamp on the right (B) shows the incorrect headlamp aim. 10. Repeat Steps 7 through 9 for the opposite headlamp. 5-57 Bulb Replacement For the proper type of replacement bulbs, see Replacement Bulbs on page 5-63. For any bulb changing procedure not listed in this section, contact your dealer/retailer. Halogen Bulbs { CAUTION: Halogen bulbs have pressurized gas inside and can burst if you drop or scratch the bulb. You or others could be injured. Be sure to read and follow the instructions on the bulb package. Headlamps To replace one of these bulbs: 1. Open the hood. See Hood Release on page 5-13 for more information. 2. If you are replacing the bulb on the passenger side remove the Engine Air Cleaner cover. See Engine Air Cleaner/Filter on page 5-19 for more information. 3. Reach in and access the bulb sockets from inside the engine compartment. 5-58 Center High-Mounted Stoplamp (CHMSL) and Cargo Lamp To replace one of these bulbs: 4. Turn the bulb socket counterclockwise to remove it from the headlamp assembly and pull it straight out. 5. Unplug the electrical connector from the old bulb by releasing the clips on the bulb socket. 6. Replace it with a new bulb socket. 7. Plug in the electrical connector to the new bulb socket. 8. Reinstall the new bulb socket into the headlamp assembly and turn it clockwise to secure. 9. Close the hood. 1. Remove the screws and lift off the lamp assembly. 5-59 A. Cargo Lamp B. Center High-Mounted Stoplamp Bulb (CHMSL) 2. Remove the bulbs by turning socket counterclockwise and pulling the bulb straight out. 3. Install the bulbs by pushing the bulb straight in and turning clockwise. 4. Reinstall the lamp assembly and tighten the screws. Pickup Box Identification and Fender Marker Lamps To replace a pickup box identification or fender marker lamp bulb: 1. Press the tab from the back to remove the lamp. 2. Unplug the lamp assembly harness. 3. Gently pry the individual lamp from the lamp assembly. 4. Unplug the lamp. 5. Plug in a new lamp and snap it into the assembly. 6. Reinstall the lamp assembly. 5-60 Taillamps, Turn Signal, Stoplamps and Back-up Lamps To replace one of these bulbs: 1. Open the tailgate. Tailgate on page 2-12 for more information. 2. Remove the two rear lamp assembly screws near the tailgate latch strikerpost and pull out the lamp assembly. 3. Determine which of the following taillamp assemblies applies to your vehicle. Dually Models A. Stoplamp/Taillamp/Turn Signal Lamp B. Back-up Lamp C. Stoplamp/Taillamp/Turn Signal Lamp 4. Press the tab, if the vehicle has one, and turn the bulb socket counterclockwise to remove it from the taillamp assembly. 5-61 Taillamps (Chassis Cab Models) All Other Models A. Stoplamp/Taillamp/ Turn Signal Lamp B. Stoplamp/Taillamp/ Turn Signal Lamp C. Back-up Lamp D. Sidemarker Lamp 5. Pull the old bulb straight out from the socket. 6. Press a new bulb into the socket and turn the socket clockwise into the taillamp assembly. 7. Reinstall the taillamp assembly. 5-62 A. Backup Lamp B. Stoplamp/Taillamp/Turn Signal Lamp To replace one of these bulbs: 1. Remove the four screws. 2. Lift the lens off the lamp assembly. 3. Turn the old bulb counterclockwise and pull the it straight out from the socket. 4. Install a new bulb into the socket, turn it clockwise, and press it in until it is tight. 5. Reinstall the lens and the four screws. License Plate Lamp To replace one of these bulbs: 1. Reach under the rear bumper for the bulb socket. 2. Turn the bulb socket counterclockwise and pull the bulb socket out of the connector. 3. Pull the old bulb from the bulb socket, keeping the bulb straight as you pull it out. 4. Install the new bulb. 5. Reverse Steps 1 through 3 to reinstall the bulb socket. Replacement Bulbs Exterior Lamp Back-up Lamp Back-up Lamp* Cargo Lamp and Center High-Mounted Stoplamp (CHMSL) Fender Marker Lamp High-Beam Headlamp Low-Beam Headlamp License Plate Lamp Sidemarker Lamp/Stoplamp/ Taillamp/Turn Signal Lamp Stoplamp/Turn Signal Lamp/Taillamp* * Chassis Cab Models Bulb Number 3047 1156 912 W5WLL 9005 H11 168 3047 1157 For replacement bulbs not listed here, contact your dealer/retailer. 5-63 Windshield Wiper Blade Replacement Windshield wiper blades should be inspected for wear or cracking. See Scheduled Maintenance (Gasoline Engine) on page 6-4 for more information on wiper blade inspection. Replacement blades come in different types and are removed in different ways. Here is how to remove the wiper blade: 1. Pull the windshield wiper arm connector away from the windshield. 2. Squeeze the grooved areas on each side of the blade and turn the blade assembly away from the arm connector. 3. Install the new blade onto the arm connector and make sure the grooved areas are fully set in the locked position. For the proper type and size, see Maintenance Replacement Parts on page 6-17. Tires Your new vehicle comes with high-quality tires made by a leading tire manufacturer. If you ever have questions about your tire warranty and where to obtain service, see your vehicle Warranty booklet for details. For additional information refer to the tire manufacturer. 5-64 { CAUTION: Poorly maintained and improperly used tires are dangerous. · Overloading your vehicle's tires can cause overheating as a result of too much flexing. You could have an air-out and a serious accident. See Loading the Vehicle on page 4-32. · Underinflated tires pose the same danger as overloaded tires. The resulting accident could cause serious injury. Check all tires frequently to maintain the recommended pressure. Tire pressure should be checked when your vehicle's tires are cold. See Inflation - Tire Pressure on page 5-73. · Overinflated tires are more likely to be cut, punctured, or broken by a sudden impact -- such as when you hit a pothole. Keep tires at the recommended pressure. · Worn, old tires can cause accidents. If the tire's tread is badly worn, or if your vehicle's tires have been damaged, replace them. 20-Inch Tires If your vehicle has the optional 20-inch P275/55R20 size tires, they are classified as touring tires and are designed for on road use. The low-profile, wide tread design is not recommended for "off-road" driving or commercial uses such as snow plowing. See Off-Road Driving on page 4-12 and Adding a Snow Plow or Similar Equipment on page 4-38 for additional information. Notice: If the vehicle has low-profile tires, they are more susceptible to damage from road hazards or curb impact than standard profile tires. Tire and/or wheel assembly damage can occur when coming into contact with road hazards like, potholes, or sharp edged objects, or when sliding into a curb. The vehicle warranty does not cover this type of damage. Keep tires set to the correct inflation pressure and, when possible avoid contact with curbs, potholes, and other road hazards. 5-65 Tire Sidewall Labeling Useful information about a tire is molded into the sidewall. The following illustrations are examples of a typical P-Metric and a LT-Metric tire sidewall. Passenger (P-Metric) Tire (A) Tire Size: The tire size code is a combination of letters and numbers used to define a particular tire's width, height, aspect ratio, construction type, and service description. See the "Tire Size" illustration later in this section for more detail. 5-66 (B) TPC Spec (Tire Performance Criteria Specification): Original equipment tires designed to GM's specific tire performance criteria have a TPC specification code molded onto the sidewall. GM's TPC specifications meet or exceed all federal safety guidelines. (C) DOT (Department of Transportation): The Department of Transportation (DOT) code indicates that the tire is in compliance with the U.S. Department of Transportation Motor Vehicle Safety Standards. (D) Tire Identification Number (TIN): The letters and numbers following DOT code are the Tire Identification Number (TIN). The TIN shows the manufacturer and plant code, tire size, and date the tire was manufactured. The TIN is molded onto both sides of the tire, although only one side may have the date of manufacture. (E) Tire Ply Material: The type of cord and number of plies in the sidewall and under the tread. (F) Uniform Tire Quality Grading (UTQG): Tire manufacturers are required to grade tires based on three performance factors: treadwear, traction, and temperature resistance. For more information, see Uniform Tire Quality Grading on page 5-86. (G) Maximum Cold Inflation Load Limit: Maximum load that can be carried and the maximum pressure needed to support that load. For information on recommended tire pressure see Inflation - Tire Pressure on page 5-73 and Loading the Vehicle on page 4-32. Light Truck (LT-Metric) Tire (A) Tire Size: The tire size code is a combination of letters and numbers used to define a particular tire's width, height, aspect ratio, construction type, and service description. See the "Tire Size" illustration later in this section for more detail. (B) TPC Spec (Tire Performance Criteria Specification): Original equipment tires designed to GM's specific tire performance criteria have a TPC specification code molded onto the sidewall. GM's TPC specifications meet or exceed all federal safety guidelines. (C) Dual Tire Maximum Load: Maximum load that can be carried and the maximum pressure needed to support that load when used in a dual configuration. For information on recommended tire pressure see Inflation - Tire Pressure on page 5-73 and Loading the Vehicle on page 4-32. (D) DOT (Department of Transportation): The Department of Transportation (DOT) code indicates that the tire is in compliance with the U.S. Department of Transportation Motor Vehicle Safety Standards. 5-67 (E) Tire Identification Number (TIN): The letters and numbers following DOT code are the Tire Identification Number (TIN). The TIN shows the manufacturer and plant code, tire size, and date the tire was manufactured. The TIN is molded onto both sides of the tire, although only one side may have the date of manufacture. (F) Tire Ply Material: The type of cord and number of plies in the sidewall and under the tread. (G) Single Tire Maximum Load: Maximum load that can be carried and the maximum pressure needed to support that load when used as a single. For information on recommended tire pressure see Inflation - Tire Pressure on page 5-73 and Loading the Vehicle on page 4-32. Tire Size The following examples show the different parts of a tire size. Passenger (P-Metric) Tire (A) Passenger (P-Metric) Tire: The United States version of a metric tire sizing system. The letter P as the first character in the tire size means a passenger vehicle tire engineered to standards set by the U.S. Tire and Rim Association. (B) Tire Width: The three-digit number indicates the tire section width in millimeters from sidewall to sidewall. 5-68 (C) Aspect Ratio: A two-digit number that indicates the tire height-to-width measurements. For example, if the tire size aspect ratio is 75, as shown in item C of the tire illustration, it would mean that the tire's sidewall is 75 percent as high as it is wide. (D) Construction Code: A letter code is used to indicate the type of ply construction in the tire. The letter R means radial ply construction; the letter D means diagonal or bias ply construction; and the letter B means belted-bias ply construction. (E) Rim Diameter: Diameter of the wheel in inches. (F) Service Description: These characters represent the load index and speed rating of the tire. The load index represents the load carry capacity a tire is certified to carry. The speed rating is the maximum speed a tire is certified to carry a load. Light Truck (LT-Metric) Tire (A) Light Truck (LT-Metric) Tire: The United States version of a metric tire sizing system. The letters LT as the first two characters in the tire size means a light truck tire engineered to standards set by the U.S. Tire and Rim Association. (B) Tire Width: The three-digit number indicates the tire section width in millimeters from sidewall to sidewall. (C) Aspect Ratio: A two-digit number that indicates the tire height-to-width measurements. For example, if the tire size aspect ratio is 75, as shown in item C of the light truck (LT-Metric) tire illustration, it would mean that the tire's sidewall is 75 percent as high as it is wide. 5-69 (D) Construction Code: A letter code is used to indicate the type of ply construction in the tire. The letter R means radial ply construction; the letter D means diagonal or bias ply construction; and the letter B means belted-bias ply construction. (E) Rim Diameter: Diameter of the wheel in inches. (F) Load Range : Load Range. (G) Service Description: The service description indicates the load index and speed rating of a tire. If two numbers are given as in the example, 120/116, then this represents the load index for single versus dual wheel usage (single/dual). The speed rating is the maximum speed a tire is certified to carry a load. Tire Terminology and Definitions Air Pressure: The amount of air inside the tire pressing outward on each square inch of the tire. Air pressure is expressed in pounds per square inch (psi) or kilopascal (kPa). Accessory Weight: This means the combined weight of optional accessories. Some examples of optional accessories are, automatic transmission, power steering, power brakes, power windows, power seats, and air conditioning. 5-70 Aspect Ratio: The relationship of a tire's height to its width. Belt: A rubber coated layer of cords that is located between the plies and the tread. Cords may be made from steel or other reinforcing materials. Bead: The tire bead contains steel wires wrapped by steel cords that hold the tire onto the rim. Bias Ply Tire: A pneumatic tire in which the plies are laid at alternate angles less than 90 degrees to the centerline of the tread. Cold Tire Pressure: The amount of air pressure in a tire, measured in pounds per square inch (psi) or kilopascals (kPa) before a tire has built up heat from driving. See Inflation - Tire Pressure on page 5-73. Curb Weight: The weight of a motor vehicle with standard and optional equipment including the maximum capacity of fuel, oil, and coolant, but without passengers and cargo. DOT Markings: A code molded into the sidewall of a tire signifying that the tire is in compliance with the U.S. Department of Transportation (DOT) motor vehicle safety standards. The DOT code includes the Tire Identification Number (TIN), an alphanumeric designator which can also identify the tire manufacturer, production plant, brand, and date of production. GVWR: Gross Vehicle Weight Rating. See Loading the Vehicle on page 4-32. GAWR FRT: Gross Axle Weight Rating for the front axle. See Loading the Vehicle on page 4-32. GAWR RR: Gross Axle Weight Rating for the rear axle. See Loading the Vehicle on page 4-32. Intended Outboard Sidewall: The side of an asymmetrical tire, that must always face outward when mounted on a vehicle. Kilopascal (kPa): The metric unit for air pressure. Light Truck (LT-Metric) Tire: A tire used on light duty trucks and some multipurpose passenger vehicles. Load Index: An assigned number ranging from 1 to 279 that corresponds to the load carrying capacity of a tire. Maximum Inflation Pressure: The maximum air pressure to which a cold tire can be inflated. The maximum air pressure is molded onto the sidewall. Maximum Load Rating: The load rating for a tire at the maximum permissible inflation pressure for that tire. Maximum Loaded Vehicle Weight: The sum of curb weight, accessory weight, vehicle capacity weight, and production options weight. Normal Occupant Weight: The number of occupants a vehicle is designed to seat multiplied by 150 lbs (68 kg). See Loading the Vehicle on page 4-32. Occupant Distribution: Designated seating positions. Outward Facing Sidewall: The side of an asymmetrical tire that has a particular side that faces outward when mounted on a vehicle. The side of the tire that contains a whitewall, bears white lettering, or bears manufacturer, brand, and/or model name molding that is higher or deeper than the same moldings on the other sidewall of the tire. Passenger (P-Metric) Tire: A tire used on passenger cars and some light duty trucks and multipurpose vehicles. Recommended Inflation Pressure: Vehicle manufacturer's recommended tire inflation pressure as shown on the tire placard. See Inflation - Tire Pressure on page 5-73 and Loading the Vehicle on page 4-32. 5-71 Radial Ply Tire: A pneumatic tire in which the ply cords that extend to the beads are laid at 90 degrees to the centerline of the tread. Rim: A metal support for a tire and upon which the tire beads are seated. Sidewall: The portion of a tire between the tread and the bead. Speed Rating: An alphanumeric code assigned to a tire indicating the maximum speed at which a tire can operate. Traction: The friction between the tire and the road surface. The amount of grip provided. Tread: The portion of a tire that comes into contact with the road. Treadwear Indicators: Narrow bands, sometimes called wear bars, that show across the tread of a tire when only 1/16 inch (1.6 mm) of tread remains. See When It Is Time for New Tires on page 5-83. UTQGS (Uniform Tire Quality Grading Standards): A tire information system that provides consumers with ratings for a tire's traction, temperature, and treadwear. Ratings are determined by tire manufacturers using government testing procedures. The ratings are molded into the sidewall of the tire. See Uniform Tire Quality Grading on page 5-86. Vehicle Capacity Weight: The number of designated seating positions multiplied by 150 lbs (68 kg) plus the rated cargo load. See Loading the Vehicle on page 4-32. Vehicle Maximum Load on the Tire: Load on an individual tire due to curb weight, accessory weight, occupant weight, and cargo weight. Vehicle Placard: A label permanently attached to a vehicle showing the vehicle's capacity weight and the original equipment tire size and recommended inflation pressure. See "Tire and Loading Information Label" under Loading the Vehicle on page 4-32. 5-72 Inflation - Tire Pressure Tires need the correct amount of air pressure to operate effectively. Notice: Do not let anyone tell you that under-inflation or over-inflation is all right. It is not. If your tires do not have enough air (under-inflation), you can get the following: · Too much flexing · Too much heat · Tire overloading · Premature or irregular wear · Poor handling · Reduced fuel economy If your tires have too much air (over-inflation), you can get the following: · Unusual wear · Poor handling · Rough ride · Needless damage from road hazards A vehicle specific Tire and Loading Information label is attached to your vehicle. This label shows your vehicle's original equipment tires and the correct inflation pressures for your tires when they are cold. The recommended cold tire inflation pressure, shown on the label, is the minimum amount of air pressure needed to support your vehicle's maximum load carrying capacity. For additional information regarding how much weight your vehicle can carry, and an example of the Tire and Loading Information label, see Loading the Vehicle on page 4-32. How you load your vehicle affects vehicle handling and ride comfort. Never load your vehicle with more weight than it was designed to carry. When to Check Check your tires once a month or more. Do not forget to check the pressure of the spare tire, if your vehicle has one. See Spare Tire on page 5-112 for additional information. 5-73 How to Check Use a good quality pocket-type gage to check tire pressure. You cannot tell if your tires are properly inflated simply by looking at them. Radial tires may look properly inflated even when they're underinflated. Check the tire's inflation pressure when the tires are cold. Cold means your vehicle has been sitting for at least three hours or driven no more than 1 mile (1.6 km). Remove the valve cap from the tire valve stem. Press the tire gage firmly onto the valve to get a pressure measurement. If the cold tire inflation pressure matches the recommended pressure on the tire and loading information label, no further adjustment is necessary. If the pressure is low, add air until you reach the recommended amount. If you overfill the tire, release air by pushing on the metal stem in the center of the tire valve. Recheck the tire pressure with the tire gage. Be sure to put the valve caps back on the valve stems. They help prevent leaks by keeping out dirt and moisture. 5-74 High-Speed Operation { CAUTION: Driving at high speeds, 100 mph (160 km/h) or higher, puts an additional strain on tires. Sustained high-speed driving causes excessive heat build up and can cause sudden tire failure. You could have a crash and you or others could be killed. Some high-speed rated tires require inflation pressure adjustment for high speed operation. When speed limits and road conditions are such that a vehicle can be driven at high speeds, make sure the tires are rated for high speed operation, in excellent condition, and set to the correct cold tire inflation pressure for the vehicle load. Vehicles with P265/70R17 or P275/55R20 size tires require inflation pressure adjustment when driving the vehicle at speeds of 100 mph (160 km/h) or higher. Set the cold tire inflation pressure to 3 psi (20 kPa) above the recommended cold tire pressure shown on the Tire and Loading Information label. When you end this high-speed driving, return the tires to the cold inflation pressure shown on the Tire and Loading Information label. See Loading the Vehicle on page 4-32 and Inflation - Tire Pressure on page 5-73. Dual Tire Operation When the vehicle is new, or whenever a wheel, wheel bolt or wheel nut is replaced, check the wheel nut torque after 100, 1,000 and 6,000 miles (160, 1 600 and 10 000 km) of driving. For proper torque and wheel nut tightening information, see Removing the Spare Tire and Tools on page 5-93. The outer tire on a dual wheel setup generally wears faster than the inner tire. Your tires will wear more evenly and last longer if you rotate the tires periodically, see Tire Inspection and Rotation on page 5-80. Also see Scheduled Maintenance (Gasoline Engine) on page 6-4. { CAUTION: If you operate your vehicle with a tire that is badly underinflated, the tire can overheat. An overheated tire can lose air suddenly or catch fire. You or others could be injured. Be sure all tires (including the spare) are properly inflated. See Inflation - Tire Pressure on page 5-73, for information on proper tire inflation. Tire Pressure Monitor System The Tire Pressure Monitor System (TPMS) uses radio and sensor technology to check tire pressure levels. The TPMS sensors monitor the air pressure in your vehicle's tires and transmit tire pressure readings to a receiver located in the vehicle. Each tire, including the spare (if provided), should be checked monthly when cold and inflated to the inflation pressure recommended by the vehicle manufacturer on the vehicle placard or tire inflation pressure label. (If your vehicle has tires of a different size than the size indicated on the vehicle placard or tire inflation pressure label, you should determine the proper tire inflation pressure for those tires.) As an added safety feature, your vehicle has been equipped with a tire pressure monitoring system (TPMS) that illuminates a low tire pressure telltale when one or more of your tires is significantly under-inflated. Accordingly, when the low tire pressure telltale illuminates, you should stop and check your tires as soon as possible, and inflate them to the proper pressure. Driving on a significantly under-inflated tire causes the tire to overheat and can lead to tire failure. Under-inflation also reduces fuel efficiency and tire tread life, and may affect the vehicle's handling and stopping ability. 5-75 Please note that the TPMS is not a substitute for proper tire maintenance, and it is the driver's responsibility to maintain correct tire pressure, even if under-inflation has not reached the level to trigger illumination of the TPMS low tire pressure telltale. Your vehicle has also been equipped with a TPMS malfunction indicator to indicate when the system is not operating properly. The TPMS malfunction indicator is combined with the low tire pressure telltale. When the system detects a malfunction, the telltale will flash for approximately one minute and then remain continuously illuminated. This sequence will continue upon subsequent vehicle start-ups as long as the malfunction exists. When the malfunction indicator is illuminated, the system may not be able to detect or signal low tire pressure as intended. TPMS malfunctions may occur for a variety of reasons, including the installation of replacement or alternate tires or wheels on the vehicle that prevent the TPMS from functioning properly. Always check the TPMS malfunction telltale after replacing one or more tires or wheels on your vehicle to ensure that the replacement or alternate tires and wheels allow the TPMS to continue to function properly. See Tire Pressure Monitor Operation on page 5-77 for additional information. Federal Communications Commission (FCC) and Industry and Science Canada The Tire Pressure Monitor System (TPMS) operates on a radio frequency and complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: 1. This device may not cause harmful interference. 2. This device must accept any interference received, including interference that may cause undesired operation. Vehicles with TPMS operate on a radio frequency and comply with RSS-210 of Industry and Science Canada. Operation is subject to the following two conditions: 1. This device may not cause interference. 2. This device must accept any interference received, including interference that may cause undesired operation of the device. Changes or modifications to this system by other than an authorized service facility could void authorization to use this equipment. 5-76 Tire Pressure Monitor Operation This vehicle may have a Tire Pressure Monitor System (TPMS). The TPMS is designed to warn the driver when a low tire pressure condition exists. TPMS sensors are mounted onto each tire and wheel assembly, excluding the spare tire and wheel assembly. The TPMS sensors monitor the air pressure in the vehicle's tires and transmits the tire pressure readings to a receiver located in the vehicle. When a low tire pressure condition is detected, the TPMS will illuminate the low tire pressure warning symbol located on the instrument panel cluster. At the same time a message to check the pressure in a specific tire appears on the Driver Information Center (DIC) display. The low tire pressure warning light and the DIC warning message come on at each ignition cycle until the tires are inflated to the correct inflation pressure. If your vehicle has DIC buttons, tire pressure levels can be viewed by the driver. For additional information and details about the DIC operation and displays see DIC Operation and Displays (With DIC Buttons) on page 3-53 or DIC Operation and Displays (Without DIC Buttons) on page 3-59 and DIC Warnings and Messages on page 3-66. The low tire pressure warning light may come on in cool weather when the vehicle is first started, and then turn off as you start to drive. This could be an early indicator that the air pressure in the tire(s) are getting low and need to be inflated to the proper pressure. A Tire and Loading Information label, attached to your vehicle, shows the size of your vehicle's original equipment tires and the correct inflation pressure for your vehicle's tires when they are cold. See Loading the Vehicle on page 4-32, for an example of the Tire and Loading Information label and its location on your vehicle. Also see Inflation - Tire Pressure on page 5-73. Your vehicle's TPMS can warn you about a low tire pressure condition but it does not replace normal tire maintenance. See Tire Inspection and Rotation on page 5-80 and Tires on page 5-64. Notice: Liquid tire sealants could damage the Tire Pressure Monitor System (TPMS) sensors. Sensor damage caused by using a tire sealant is not covered by your warranty. Do not use liquid tire sealants. 5-77 TPMS Malfunction Light and Message The TPMS will not function properly if one or more of the TPMS sensors are missing or inoperable. When the system detects a malfunction, the low tire warning light flashes for about one minute and then stays on for the remainder of the ignition cycle. A DIC warning message is also displayed. The low tire warning light and DIC warning message come on at each ignition cycle until the problem is corrected. Some of the conditions that can cause the malfunction light and DIC message to come on are: · One of the road tires has been replaced with the spare tire. The spare tire does not have a TPMS sensor. The TPMS malfunction light and DIC message should go off once you re-install the road tire containing the TPMS sensor. · The TPMS sensor matching process was started but not completed or not completed successfully after rotating the vehicle's tires. The DIC message and TPMS malfunction light should go off once the TPMS sensor matching process is performed successfully. See "TPMS Sensor Matching Process" later in this section. · One or more TPMS sensors are missing or damaged. The DIC message and the TPMS malfunction light should go off when the TPMS sensors are installed and the sensor matching process is performed successfully. See your dealer/ retailer for service. · Replacement tires or wheels do not match your vehicle's original equipment tires or wheels. Tires and wheels other than those recommended for your vehicle could prevent the TPMS from functioning properly. See Buying New Tires on page 5-84. · Operating electronic devices or being near facilities using radio wave frequencies similar to the TPMS could cause the TPMS sensors to malfunction. If the TPMS is not functioning it cannot detect or signal a low tire condition. See your dealer/retailer for service if the TPMS malfunction light and DIC message comes on and stays on. 5-78 TPMS Sensor Matching Process Each TPMS sensor has a unique identification code. Any time you rotate your vehicle's tires or replace one or more of the TPMS sensors, the identification codes will need to be matched to the new tire/wheel position. The sensors are matched to the tire/wheel positions in the following order: driver side front tire, passenger side front tire, passenger side rear tire, and driver side rear tire using a TPMS diagnostic tool. See your dealer/retailer for service. The TPMS sensors can also be matched to each tire/wheel position by increasing or decreasing the tire's air pressure. If increasing the tire's air pressure, do not exceed the maximum inflation pressure indicated on the tire's sidewall. To decrease air-pressure out of a tire you can use the pointed end of the valve cap, a pencil-style air pressure gage, or a key. You have two minutes to match the first tire/wheel position, and five minutes overall to match all four tire/wheel positions. If it takes longer than two minutes, to match the first tire and wheel, or more than five minutes to match all four tire and wheel positions the matching process stops and you need to start over. The TPMS sensor matching process is outlined below: 1. Set the parking brake. 2. Turn the ignition switch to ON/RUN with the engine off. 3. Press the Remote Keyless Entry (RKE) transmitter's LOCK and UNLOCK buttons at the same time for approximately five seconds. The horn sounds twice to signal the receiver is in relearn mode and TIRE LEARNING ACTIVE message displays on the DIC screen. If your vehicle does not have RKE, press the Driver Information Center (DIC) vehicle information button until the PRESS V TO RELEARN TIRE POSITIONS message displays. The horn sounds twice to signal the receiver is in relearn mode and TIRE LEARNING ACTIVE message displays on the DIC screen. If your vehicle does not have RKE or DIC buttons, press the trip odometer reset stem located on the instrument panel cluster until the PRESS V TO RELEARN TIRE POSITIONS message displays. The horn sounds twice to signal the receiver is in relearn mode and TIRE LEARNING ACTIVE message displays on the DIC screen. 4. Start with the driver side front tire. 5-79 5. Remove the valve cap from the valve cap stem. Activate the TPMS sensor by increasing or decreasing the tire's air pressure for five seconds, or until a horn chirp sounds. The horn chirp, which may take up to 30 seconds to sound, confirms that the sensor identification code has been matched to this tire and wheel position. 6. Proceed to the passenger side front tire, and repeat the procedure in Step 5. 7. Proceed to the passenger side rear tire, and repeat the procedure in Step 5. 8. Proceed to the driver side rear tire, and repeat the procedure in Step 5. The horn sounds two times to indicate the sensor identification code has been matched to the driver side rear tire, and that the TPMS sensor matching process is no longer active. The TIRE LEARNING ACTIVE message on the DIC display screen goes off. 9. Turn the ignition switch to LOCK/OFF. 10. Set all four tires to the recommended air pressure level as indicated on the Tire and Loading Information label. 11. Put the valve caps back on the valve stems. Tire Inspection and Rotation We recommend that you regularly inspect your vehicle's tires, including the spare tire, for signs of wear or damage. See When It Is Time for New Tires on page 5-83 for more information. Tires should be rotated every 5,000 to 8,000 miles (8 000 to 13 000 km). See Scheduled Maintenance (Gasoline Engine) on page 6-4. The purpose of a regular tire rotation is to achieve a uniform wear for all tires on the vehicle. This will ensure that your vehicle continues to perform most like it did when the tires were new. Any time you notice unusual wear, rotate your tires as soon as possible and check wheel alignment. Also check for damaged tires or wheels. See When It Is Time for New Tires on page 5-83 and Wheel Replacement on page 5-88. If your vehicle has dual rear wheels, also see Dual Tire Operation on page 5-75. 5-80 If your vehicle has single rear wheels and the tread design for the front tires is the same as the rear tires, use the rotation pattern shown here when rotating the tires. If your vehicle has dual rear wheels and the tread design for the front tires is the same as the rear tires, always use one of the correct rotation patterns shown here when rotating the tires. 5-81 If your vehicle has dual rear wheels and the tread design for the front tires is different from the dual rear tires, always use the correct rotation pattern shown here when rotating the tires. The dual tires are rotated as a pair, and the inside rear tires become the outside rear tires. When you install dual wheels, be sure the vent holes in the inner and outer wheels on each side are lined up. After the tires have been rotated, adjust the front and rear inflation pressures as shown on the Tire and Loading Information label. See Inflation - Tire Pressure on page 5-73 and Loading the Vehicle on page 4-32. Make certain that all wheel nuts are properly tightened. See "Wheel Nut Torque" under Capacities and Specifications on page 5-129. { CAUTION: Rust or dirt on a wheel, or on the parts to which it is fastened, can make wheel nuts become loose after time. The wheel could come off and cause an accident. When you change a wheel, remove any rust or dirt from places where the wheel attaches to the vehicle. In an emergency, you can use a cloth or a paper towel to do this; but be sure to use a scraper or wire brush later, if needed, to get all the rust or dirt off. See Changing a Flat Tire on page 5-92. If your vehicle has a Tire Pressure Monitor System (TPMS), reset the TPMS sensors after rotating the tires. See Tire Pressure Monitor Operation on page 5-77. Make sure the spare tire, if your vehicle has one, is stored securely. Push, pull, and then try to rotate or turn the tire. If it moves, tighten the cable. See Storing a Flat or Spare Tire and Tools on page 5-108. 5-82 When It Is Time for New Tires Various factors, such as maintenance, temperatures, driving speeds, vehicle loading, and road conditions, influence when you need new tires. One way to tell when it is time for new tires is to check the treadwear indicators, which appear when your tires have only 1/16 inch (1.6 mm) or less of tread remaining. Some commercial truck tires may not have treadwear indicators. You need new tires if any of the following statements are true: · You can see the indicators at three or more places around the tire. · You can see cord or fabric showing through the tire's rubber. · The tread or sidewall is cracked, cut, or snagged deep enough to show cord or fabric. · The tire has a bump, bulge, or split. · The tire has a puncture, cut, or other damage that cannot be repaired well because of the size or location of the damage. The rubber in tires degrades over time, even if they are not being used. This is also true for the spare tire, if your vehicle has one. Multiple conditions affect how fast this aging takes place, including temperatures, loading conditions, and inflation pressure maintenance. With proper care and maintenance tires will typically wear out before they degrade due to age. If you are unsure about the need to replace your tires as they get older, consult the tire manufacturer for more information. 5-83 Buying New Tires GM has developed and matched specific tires for your vehicle. The original equipment tires installed on your vehicle, when it was new, were designed to meet General Motors Tire Performance Criteria Specification (TPC spec) system rating. If you need replacement tires, GM strongly recommends that you get tires with the same TPC Spec rating. This way, your vehicle will continue to have tires that are designed to give the same performance and vehicle safety, during normal use, as the original tires. GM's exclusive TPC Spec system considers over a dozen critical specifications that impact the overall performance of your vehicle, including brake system performance, ride and handling, traction control, and tire pressure monitoring performance. GM's TPC Spec number is molded onto the tire's sidewall near the tire size. If the tires have an all-season tread design, the TPC spec number will be followed by a MS, for mud and snow. See Tire Sidewall Labeling on page 5-66 for additional information. GM recommends replacing tires in sets of four (or six if your vehicle has dual rear wheels). This is because uniform tread depth on all tires will help keep your vehicle performing most like it did when the tires were new. Replacing less than a full set of tires can affect the braking and handling performance of your vehicle. See Tire Inspection and Rotation on page 5-80 for information on proper tire rotation. { CAUTION: Mixing tires could cause you to lose control while driving. If you mix tires of different sizes, brands, or types (radial and bias-belted tires) the vehicle may not handle properly, and you could have a crash. Using tires of different sizes, brands, or types may also cause damage to your vehicle. Be sure to use the correct size, brand, and type of tires on your vehicle's wheels. 5-84 { CAUTION: If you use bias-ply tires on the vehicle, the wheel rim flanges could develop cracks after many miles of driving. A tire and/or wheel could fail suddenly, causing a crash. Use only radial-ply tires with the wheels on the vehicle. If you must replace your vehicle's tires with those that do not have a TPC Spec number, make sure they are the same size, load range, speed rating, and construction type (radial and bias-belted tires) as your vehicle's original tires. Vehicles that have a tire pressure monitoring system could give an inaccurate low-pressure warning if non-TPC Spec rated tires are installed on your vehicle. Non-TPC Spec rated tires may give a low-pressure warning that is higher or lower than the proper warning level you would get with TPC Spec rated tires. See Tire Pressure Monitor System on page 5-75. Your vehicle's original equipment tires are listed on the Tire and Loading Information label. See Loading the Vehicle on page 4-32, for more information about the Tire and Loading Information label and its location on your vehicle. 5-85 Different Size Tires and Wheels If you add wheels or tires that are a different size than your original equipment wheels and tires, this could affect the way your vehicle performs, including its braking, ride and handling characteristics, stability, and resistance to rollover. Additionally, if your vehicle has electronic systems such as anti-lock brakes, rollover airbags, traction control, and electronic stability control, the performance of these systems can be affected. { CAUTION: If you add different sized wheels, your vehicle may not provide an acceptable level of performance and safety if tires not recommended for those wheels are selected. You may increase the chance that you will crash and suffer serious injury. Only use GM specific wheel and tire systems developed for your vehicle, and have them properly installed by a GM certified technician. See Buying New Tires on page 5-84 and Accessories and Modifications on page 5-4 for additional information. Uniform Tire Quality Grading Quality grades can be found where applicable on the tire sidewall between tread shoulder and maximum section width. For example: Treadwear 200 Traction AA Temperature A The following information relates to the system developed by the United States National Highway Traffic Safety Administration (NHTSA), which grades tires by treadwear, traction, and temperature performance. This applies only to vehicles sold in the United States. The grades are molded on the sidewalls of most passenger car tires. The Uniform Tire Quality Grading (UTQG) system does not apply to deep tread, winter-type snow tires, space-saver, or temporary use spare tires, tires with nominal rim diameters of 10 to 12 inches (25 to 30 cm), or to some limited-production tires. While the tires available on General Motors passenger cars and light trucks may vary with respect to these grades, they must also conform to federal safety requirements and additional General Motors Tire Performance Criteria (TPC) standards. 5-86 Treadwear The treadwear grade is a comparative rating based on the wear rate of the tire when tested under controlled conditions on a specified government test course. For example, a tire graded 150 would wear one and a half (1.5) times as well on the government course as a tire graded 100. The relative performance of tires depends upon the actual conditions of their use, however, and may depart significantly from the norm due to variations in driving habits, service practices, and differences in road characteristics and climate. Traction AA, A, B, C The traction grades, from highest to lowest, are AA, A, B, and C. Those grades represent the tire's ability to stop on wet pavement as measured under controlled conditions on specified government test surfaces of asphalt and concrete. A tire marked C may have poor traction performance. Temperature A, B, C The temperature grades are A (the highest), B, and C, representing the tire's resistance to the generation of heat and its ability to dissipate heat when tested under controlled conditions on a specified indoor laboratory test wheel. Sustained high temperature can cause the material of the tire to degenerate and reduce tire life, and excessive temperature can lead to sudden tire failure. The grade C corresponds to a level of performance which all passenger car tires must meet under the Federal Motor Vehicle Safety Standard No. 109. Grades B and A represent higher levels of performance on the laboratory test wheel than the minimum required by law. It should be noted that the temperature grade for this tire is established for a tire that is properly inflated and not overloaded. Excessive speed, underinflation, or excessive loading, either separately or in combination, can cause heat buildup and possible tire failure. 5-87 Wheel Alignment and Tire Balance The tires and wheels on your vehicle were aligned and balanced carefully at the factory to give you the longest tire life and best overall performance. Adjustments to wheel alignment and tire balancing will not be necessary on a regular basis. However, if you notice unusual tire wear or your vehicle pulling to one side or the other, the alignment might need to be checked. If you notice your vehicle vibrating when driving on a smooth road, the tires and wheels might need to be rebalanced. See your dealer/retailer for proper diagnosis. Wheel Replacement Replace any wheel that is bent, cracked, or badly rusted or corroded. If wheel nuts keep coming loose, the wheel, wheel bolts and wheel nuts should be replaced. If the wheel leaks air, replace it (except some aluminum wheels, which can sometimes be repaired). See your dealer/retailer if any of these conditions exist. Your dealer/retailer will know the kind of wheel you need. Each new wheel should have the same load-carrying capacity, diameter, width, offset and be mounted the same way as the one it replaces. If you need to replace any of the wheels, wheel bolts, wheel nuts or Tire Pressure Monitor System (TPMS) sensors, replace them only with new GM original equipment parts. This way, you will be sure to have the right wheel, wheel bolts, wheel nuts, and TPMS sensors for the vehicle. { CAUTION: Using the wrong replacement wheels, wheel bolts, or wheel nuts on your vehicle can be dangerous. It could affect the braking and handling of your vehicle, make your tires lose air and make you CAUTION: (Continued) 5-88 CAUTION: (Continued) lose control. You could have a collision in which you or others could be injured. Always use the correct wheel, wheel bolts, and wheel nuts for replacement. Notice: The wrong wheel can also cause problems with bearing life, brake cooling, speedometer or odometer calibration, headlamp aim, bumper height, vehicle ground clearance, and tire or tire chain clearance to the body and chassis. Whenever a wheel, wheel bolt or wheel nut is replaced on a dual wheel setup, check the wheel nut torque after 100, 1,000 and 6,000 miles (160, 1 600 and 10 000 km) of driving. For proper torque, see "Wheel Nut Torque" under Capacities and Specifications on page 5-129. See Changing a Flat Tire on page 5-92 for more information. Used Replacement Wheels { CAUTION: Putting a used wheel on the vehicle is dangerous. You cannot know how it has been used or how far it has been driven. It could fail suddenly and cause a crash. If you have to replace a wheel, use a new GM original equipment wheel. 5-89 Tire Chains { CAUTION: If your vehicle has dual wheels or P265/65R18, P275/55R20 or LT265/70R17 size tires, do not use tire chains. They can damage your vehicle because there is not enough clearance. Tire chains used on a vehicle without the proper amount of clearance can cause damage to the brakes, suspension, or other vehicle parts. The area damaged by the tire chains could cause you to lose control of your vehicle and you or others may be injured in a crash. Use another type of traction device only if its manufacturer recommends it for use on your vehicle and tire size combination and road conditions. Follow that manufacturer's instructions. CAUTION: (Continued) CAUTION: (Continued) To help avoid damage to your vehicle, drive slowly, readjust, or remove the device if it is contacting your vehicle, and do not spin your vehicle's wheels. If you do find traction devices that will fit, install them on the rear tires. Notice: If your vehicle does not have dual wheels and has a tire size other than P265/65R18, P275/55R20 or LT265/70R17, use tire chains only where legal and only when you must. Use chains that are the proper size for your tires. Install them on the tires of the rear axle. Do not use chains on the tires of the front axle. Tighten them as tightly as possible with the ends securely fastened. Drive slowly and follow the chain manufacturer's instructions. If you can hear the chains contacting your vehicle, stop and retighten them. If the contact continues, slow down until it stops. Driving too fast or spinning the wheels with chains on will damage your vehicle. 5-90 If a Tire Goes Flat It is unusual for a tire to blowout while you are driving, especially if you maintain your vehicle's tires properly. If air goes out of a tire, it is much more likely to leak out slowly. But if you should ever have a blowout, here are a few tips about what to expect and what to do: If a front tire fails, the flat tire creates a drag that pulls the vehicle toward that side. Take your foot off the accelerator pedal and grip the steering wheel firmly. Steer to maintain lane position, and then gently brake to a stop well out of the traffic lane. A rear blowout, particularly on a curve, acts much like a skid and may require the same correction you would use in a skid. In any rear blowout remove your foot from the accelerator pedal. Get the vehicle under control by steering the way you want the vehicle to go. It may be very bumpy and noisy, but you can still steer. Gently brake to a stop, well off the road if possible. { CAUTION: Lifting a vehicle and getting under it to do maintenance or repairs is dangerous without the appropriate safety equipment and training. If a jack is provided with the vehicle, it is designed only for changing a flat tire. If it is used for anything else, you or others could be badly injured or killed if the vehicle slips off the jack. If a jack is provided with the vehicle, only use it for changing a flat tire. If a tire goes flat, the next part shows how to use the jacking equipment to change a flat tire safely. 5-91 Changing a Flat Tire If a tire goes flat, avoid further tire and wheel damage by driving slowly to a level place. Turn on the hazard warning flashers. See Hazard Warning Flashers on page 3-8. { CAUTION: Changing a tire can be dangerous. The vehicle can slip off the jack and roll over or fall on you or other people. You and they could be badly injured or even killed. Find a level place to change your tire. To help prevent the vehicle from moving: 1. Set the parking brake firmly. 2. Put the shift lever in P (Park). 3. If you have a four-wheel-drive vehicle, be sure the transfer case is in a drive gear -- not in N (Neutral). 4. Turn off the engine and do not restart while the vehicle is raised. CAUTION: (Continued) 5-92 CAUTION: (Continued) 5. Do not allow passengers to remain in the vehicle. 6. Put the wheel blocks at the front and rear of the tire farthest away from the one being changed. That would be the tire on the other side, at the opposite end of the vehicle. When the vehicle has a flat tire (B), use the following example as a guide to assist you in the placement of wheel blocks (A). A. Wheel Block B. Flat Tire The following information explains how to use the jack and change a tire. Removing the Spare Tire and Tools Crew Cab A. Wing Nut Retaining Tool Kit B. Tool Kit C. Wheel Blocks D. Jack E. Jack Knob F. Wing Nut Retaining Wheel Blocks Regular Cab A. Wing Nut Retaining Tool Kit B. Tool Kit C. Wheel Blocks D. Jack E. Jack Knob F. Wing Nut Retaining Wheel Blocks 5-93 Extended Cab A. Wing Nut Retaining Tool Kit B. Tool Kit C. Wheel Blocks D. Jack E. Jack Knob For regular cab models, the equipment you will need is behind the passenger's seat. For extended and crew cab models, the equipment is on the shelf behind the passenger's side second row seat. 1. Turn the knob on the jack counterclockwise to lower the jack head to release the jack from its holder. 2. Remove the wheel blocks and the wheel block retainer by turning the wing nut counterclockwise. 3. Remove the wing nut used to retain the storage bag and tools by turning it counterclockwise. You will use the jack handle extensions and the wheel wrench to remove the underbody-mounted spare tire. 5-94 1. Open the spare tire lock cover on the bumper and use the ignition key rview ................................ 5-14 Coolant ...................................................... 5-29 Coolant Heater ............................................ 2-26 5 Engine (cont.) Coolant Temperature Gage ........................... 3-44 Cooling System ........................................... 5-28 Drive Belt Routing ....................................... 6-18 Exhaust ..................................................... 2-55 Fan Noise .................................................. 5-37 Fast Idle System ......................................... 2-25 Oil ............................................................. 5-15 Oil Life System ........................................... 5-18 Overheated Protection Operating Mode ........... 5-37 Overheating ................................................ 5-34 Starting ...................................................... 2-23 Entry Lighting ................................................. 3-20 Event Data Recorders ..................................... 7-17 Exit Lighting ................................................... 3-20 Extender, Safety Belt ....................................... 1-37 Exterior Lamps ............................................... 3-16 F Fast Idle System ............................................ 2-25 Filter Engine Air Cleaner ...................................... 5-19 Finish Damage ............................................. 5-120 Fixed Mast Antenna ....................................... 3-142 Flashers, Hazard Warning .................................. 3-8 Flash-to-Pass ................................................. 3-10 Flat Tire ........................................................ 5-91 Flat Tire, Changing ......................................... 5-92 Flat Tire, Storing ........................................... 5-108 Fluid Automatic Transmission ........................ 5-21, 5-24 Power Steering ........................................... 5-38 Windshield Washer ...................................... 5-39 Fog Lamp Fog ........................................................... 3-19 Fog Lamp Light .............................................. 3-50 Four-Wheel Drive .................................... 2-36, 5-50 Four-Wheel-Drive Light .................................... 3-51 Front Axle ...................................................... 5-52 Fuel ............................................................... 5-6 Additives ...................................................... 5-7 California Fuel .............................................. 5-7 E85 (85% Ethanol) ........................................ 5-8 Economy Driving ........................................... 4-2 Filling a Portable Fuel Container .................... 5-12 Filling the Tank ........................................... 5-10 Fuels in Foreign Countries .............................. 5-9 Gage ......................................................... 3-51 Gasoline Octane ........................................... 5-6 Gasoline Specifications .................................. 5-7 Low Fuel Warning Light ................................ 3-52 Fuel Management, Active ................................. 2-27 6 Fuses Center Instrument Panel Fuse Block ............. 5-124 Fuses and Circuit Breakers ......................... 5-122 Instrument Panel Fuse Block ....................... 5-122 Underhood Fuse Block ............................... 5-125 Windshield Wiper ....................................... 5-122 G Gage Oil Pressure ............................................... 3-48 Speedometer .............................................. 3-35 Tachometer ................................................. 3-35 Voltmeter Gage ........................................... 3-41 Gages Engine Coolant Temperature ......................... 3-44 Fuel .......................................................... 3-51 Trip Odometer ............................................. 3-35 Garage Door Opener ....................................... 2-74 Gasoline Octane ........................................................ 5-6 Specifications ............................................... 5-7 Glove Box ..................................................... 2-81 GM Mobility Reimbursement Program .................. 7-7 H Halogen Bulbs ................................................ 5-58 Hazard Warning Flashers ................................... 3-8 Head Restraints ............................................... 1-3 Headlamp Aiming ....................................................... 5-55 Headlamps .................................................... 5-58 Bulb Replacement ....................................... 5-58 Daytime Running Lamps (DRL) ..................... 3-17 Exterior Lamps ............................................ 3-16 Flash-to-Pass .............................................. 3-10 High/Low Beam Changer .............................. 3-10 On Reminder .............................................. 3-17 Pickup Box Identification and Fender Marker Lamps ......................................... 5-60 Heated Seats ................................................... 1-8 Heater ................................................... 3-24, 3-26 Engine Coolant ........................................... 2-26 Highbeam On Light ......................................... 3-50 High-Speed Operation, Tires ............................. 5-74 Highway Hypnosis ........................................... 4-26 Hill and Mountain Roads .................................. 4-27 Hood Checking Things Under ................................ 5-12 Release ..................................................... 5-13 Horn ............................................................... 3-8 How to Wear Safety Belts Properly ................... 1-21 7 I Ignition Positions ............................................. 2-21 Infants and Young Children, Restraints ............... 1-41 Inflation - Tire Pressure ................................... 5-73 Instrument Panel Brightness .................................................. 3-20 Cluster ....................................................... 3-34 Overview ............................................... 3-4, 3-6 Storage Area .............................................. 2-81 Introduction ...................................................... 6-2 J Jump Starting ................................................. 5-44 K Keyless Entry, Remote Operation ........................ 2-4 Keyless Entry System ....................................... 2-4 Keys ............................................................... 2-3 L Labeling, Tire Sidewall ..................................... 5-66 Lamp Malfunction Indicator .................................... 3-45 Lamps Auxiliary Roof Mounted Lamp ........................ 3-19 Cargo ........................................................ 3-21 Daytime Running (DRL) ................................ 3-17 Dome ........................................................ 3-20 Dome Lamp Override ................................... 3-20 License Plate .............................................. 5-63 Puddle ....................................................... 3-19 Reading ..................................................... 3-21 Lap Belt ........................................................ 1-36 Lap-Shoulder Belt ........................................... 1-30 LATCH System for Child Restraints ................... 1-49 Latches, Seatback .......................................... 1-13 Lighting Entry ......................................................... 3-20 Exit ........................................................... 3-20 Lights Airbag Off .................................................. 3-38 Airbag Readiness ........................................ 3-37 Antilock Brake System (ABS) Warning ............ 3-43 8 Lights (cont.) Brake System Warning ................................. 3-42 Charging System ......................................... 3-41 Cruise Control ............................................. 3-50 Exterior Lamps ............................................ 3-16 Flash-to-Pass .............................................. 3-10 Fog Lamp .................................................. 3-50 Four-Wheel-Drive ......................................... 3-51 Highbeam On ............................................. 3-50 High/Low Beam Changer .............................. 3-10 Low Fuel Warning ....................................... 3-52 Oil Pressure ............................................... 3-49 On Reminder .............................................. 3-17 Passenger Airbag Status Indicator .................. 3-40 Safety Belt Reminders .................................. 3-36 Security ..................................................... 3-50 StabiliTrak® Indicator .................................... 3-44 Tire Pressure .............................................. 3-45 Tow/Haul Mode ........................................... 3-51 Loading Your Vehicle ....................................... 4-32 Locking Rear Axle ............................................ 4-9 Locks Delayed Locking ........................................... 2-9 Door ........................................................... 2-9 Lockout Protection ....................................... 2-11 Power Door .................................................. 2-9 Programmable Automatic Door Locks ............. 2-10 Rear Door Security Locks ............................. 2-10 Loss of Control ............................................... 4-11 Low Fuel Warning Light ................................... 3-52 Lower Anchors and Tethers for Children ............. 1-49 Luggage Carrier .............................................. 2-82 Lumbar Manual Controls ............................................ 1-6 Power Controls ............................................. 1-7 M Maintenance Footnotes ..................................................... 6-9 Maintenance Schedule Additional Required Services ........................... 6-7 At Each Fuel Fill ......................................... 6-11 At Least Once a Month ................................ 6-12 At Least Once a Year .................................. 6-12 At the First 100, 1,000 and 6,000 Miles (160, 1 600 and 10 000km) ....................... 6-11 Maintenance Record .................................... 6-19 Maintenance Replacement Parts .................... 6-17 Maintenance Requirements ............................. 6-2 Owner Checks and Services ......................... 6-11 Recommended Fluids and Lubricants ............. 6-15 Scheduled Maintenance ................................. 6-4 Using .......................................................... 6-3 Your Vehicle and the Environment ................... 6-2 Malfunction Indicator Lamp ............................... 3-45 Manual Lumbar Controls .................................... 1-6 Manual Seats ................................................... 1-4 Manual Windows ............................................ 2-15 9 Memory Seat, Mirrors, and Pedals ...................... 1-8 Message DIC Warnings and Messages ........................ 3-66 Mirrors Automatic Dimming Rearview ........................ 2-57 Manual Rearview Mirror ................................ 2-57 Outside Convex Mirror ................................. 2-61 Outside Heated Mirrors ................................ 2-61 Outside Manual Mirrors ................................ 2-58 Outside Power Foldaway Mirrors .................... 2-59 Outside Power Mirrors .................................. 2-59 Outside Towing Mirrors ................................. 2-58 Park Tilt ..................................................... 2-60 MP3 .................................................. 3-108, 3-112 N Navigation System, Privacy .............................. 7-18 Navigation/Radio System, see Navigation Manual ..................................................... 3-118 New Vehicle Break-In ...................................... 2-21 Noise Control System, Tampering ...................... 5-54 O Odometer ...................................................... 3-35 Odometer, Trip ............................................... 3-35 Off-Road ....................................................... 4-12 Driving ....................................................... 4-12 Recovery .................................................... 4-11 Oil Engine ....................................................... 5-15 Engine Oil Life System ................................. 5-18 Pressure Gage ............................................ 3-48 Pressure Light ............................................. 3-49 Older Children, Restraints ................................ 1-38 Online Owner Center ........................................ 7-5 OnStar, Privacy .............................................. 7-18 OnStar® System, see OnStar® Manual ............... 2-71 Operation, Universal Home Remote System ........ 2-75 Outlet Adjustment ............................................ 3-33 Outlets Accessory Power ......................................... 3-22 Outside Convex Mirror ............................................. 2-61 Heated Mirrors ............................................ 2-61 Manual Mirrors ............................................ 2-58 10 Outside (cont.) Power Foldaway Mirrors ............................... 2-59 Power Mirrors ............................................. 2-59 Towing Mirrors ............................................ 2-58 Overheated Engine Protection Operating Mode .......................................... 5-37 Owner Checks and Services ............................. 6-11 Owners, Canadian .............................................. iii P Paint, Damage .............................................. 5-120 Park Shifting Into ................................................ 2-52 Shifting Out of ............................................ 2-54 Park Aid ........................................................ 2-62 Park Tilt Mirrors .............................................. 2-60 Parking Assist ........................................................ 2-62 Over Things That Burn ................................. 2-54 Parking Brake ................................................ 2-50 Passenger Airbag Status Indicator ..................... 3-40 Passenger Sensing System .............................. 1-84 Passing ......................................................... 4-11 PASS-Key® III+ Electronic Immobilizer ............... 2-19 PASS-Key® III+ Electronic Immobilizer Operation ................................................... 2-19 Perchlorate Materials Requirements, California ...... 5-5 Phone Bluetooth® ................................................ 3-118 Pickup Conversion to Chassis Cab .................... 4-45 Power Door Locks .................................................. 2-9 Electrical System ....................................... 5-122 Lumbar Controls ........................................... 1-7 Retained Accessory (RAP) ............................ 2-23 Seat ............................................................ 1-5 Sliding Rear Window .................................... 2-16 Steering Fluid ............................................. 5-38 Windows .................................................... 2-15 Pregnancy, Using Safety Belts .......................... 1-36 Privacy .......................................................... 7-16 Navigation System ....................................... 7-18 OnStar ....................................................... 7-18 Radio Frequency Identification (RFID) ............. 7-18 Program Courtesy Transportation ................................ 7-10 Programmable Automatic Door Locks ................. 2-10 Proposition 65 Warning, California ....................... 5-5 Puddle Lamps ................................................ 3-19 11 R Radio Frequency Identification (RFID), Privacy .... 7-18 Radio(s) ........................................................ 3-88 Radios Navigation/Radio System, see Navigation Manual ................................................. 3-118 Reception ................................................. 3-141 Setting the Clock ......................................... 3-86 Theft-Deterrent .......................................... 3-140 RainsenseTM II Wipers ..................................... 3-11 Reading Lamps .............................................. 3-21 Rear Axle ...................................................... 5-49 Locking ........................................................ 4-9 Rear Door Security Locks ................................ 2-10 Rear Doors .................................................... 2-11 Rear Seat Armrest .......................................... 2-83 Rear Seat Audio (RSA) System ....................... 3-138 Rear Seat Entertainment System ..................... 3-129 Rear Seat Operation ............................... 1-14, 1-15 Rear Vision Camera (RVC) .............................. 2-65 Rearview Mirror, Automatic Dimming .................. 2-57 Rearview Mirrors ............................................. 2-57 Reclining Seatbacks ........................................ 1-10 Recommended Fluids and Lubricants ................. 6-15 Recovery Hooks ............................................. 4-31 Recreational Vehicle Towing ............................. 4-45 Reimbursement Program, GM Mobility ................. 7-7 Remote Keyless Entry (RKE) System .................. 2-4 Remote Keyless Entry (RKE) System, Operation ..................................................... 2-4 Remote Vehicle Start ........................................ 2-7 Removing the Flat Tire and Installing the Spare Tire .................................................. 5-98 Removing the Spare Tire and Tools ................... 5-93 Replacement Bulbs ......................................... 5-63 Replacement Parts, Maintenance ...................... 6-17 Reporting Safety Defects to General Motors ....... 7-15 Reporting Safety Defects to the Canadian Government ................................................ 7-15 Reporting Safety Defects to the U.S. Government ................................................ 7-14 Restraint System Check Checking the Restraint Systems .................... 1-92 Replacing Restraint System Parts After a Crash .......................................... 1-93 Retained Accessory Power (RAP) ...................... 2-23 Roadside Assistance Program ............................ 7-7 Rocking Your Vehicle to Get it Out .................... 4-30 Roof Sunroof ...................................................... 2-85 Running the Vehicle While Parked ..................... 2-56 12 S Safety Belt Reminders ..................................... 3-36 Safety Belts Care of .................................................... 5-116 Extender .................................................... 1-37 How to Wear Safety Belts Properly ................ 1-21 Lap Belt ..................................................... 1-36 Lap-Shoulder Belt ........................................ 1-30 Safety Belts Are for Everyone ....................... 1-16 Use During Pregnancy ................................. 1-36 Safety Defects Reporting to Canadian Government ................ 7-15 Reporting to General Motors ......................... 7-15 Reporting to the U.S. Government ................. 7-14 Safety Warnings and Symbols .............................. iv Scheduled Maintenance ..................................... 6-4 Additional Required Services ........................... 6-7 Scheduling Appointments ................................. 7-10 Seatback Latches ........................................... 1-13 Seats Center Seat ................................................ 1-13 Head Restraints ............................................ 1-3 Heated Seats ............................................... 1-8 Manual Lumbar ............................................. 1-6 Memory, Mirrors and Pedals ........................... 1-8 Power Lumbar .............................................. 1-7 Seats (cont.) Power Seats ................................................. 1-5 Rear Seat Operation ............................ 1-14, 1-15 Reclining Seatbacks ..................................... 1-10 Secondary Latch System ................................ 5-105 Securing a Child Restraint Center Front Seat Position ............................ 1-61 Rear Seat Position ...................................... 1-58 Right Front Seat Position .............. 1-62, 1-66, 1-70 Security Light ................................................. 3-50 Service ........................................................... 5-4 Accessories and Modifications ......................... 5-4 Adding Equipment to the Outside of the Vehicle ..................................................... 5-6 Doing Your Own Work ................................... 5-5 Engine Soon Lamp ...................................... 3-45 Publications Ordering Information ................... 7-15 Scheduling Appointments .............................. 7-10 Service Parts Identification Label ..................... 5-121 Servicing Your Airbag-Equipped Vehicle .............. 1-90 Setting the Clock ............................................ 3-86 Sheet Metal Damage ..................................... 5-119 Shifting Out of Park ................................................ 2-54 Shifting Into Park ............................................ 2-52 Signals, Turn and Lane-Change .......................... 3-9 Sliding Rear Window, Power ............................. 2-16 13 Snow Plow .................................................... 4-38 Spare Tire .................................................... 5-112 Installing .................................................... 5-98 Removing ................................................... 5-93 Storing ..................................................... 5-108 Specifications and Capacities .......................... 5-129 Speedometer .................................................. 3-35 StabiliTrak® Indicator Light ............................... 3-44 StabiliTrak® System .......................................... 4-6 Start Vehicle, Remote ....................................... 2-7 Starting the Engine ......................................... 2-23 Steering .......................................................... 4-9 Steering Wheel Controls, Audio ....................... 3-140 Steering Wheel, Tilt Wheel ................................. 3-8 Storage Areas Cargo Management System .......................... 2-83 Center Console ........................................... 2-82 Cupholders ................................................. 2-81 Glove Box .................................................. 2-81 Instrument Panel ......................................... 2-81 Luggage Carrier .......................................... 2-82 Rear Seat Armrest ....................................... 2-83 Stuck in Sand, Mud, Ice, or Snow ..................... 4-30 Sun Visors ..................................................... 2-17 Sunroof ......................................................... 2-85 T Tachometer .................................................... 3-35 Tailgate ......................................................... 2-12 Taillamps Turn Signal, Stoplamps and Back-up Lamps .... 5-61 Tampering with Noise Control System Prohibited ................................................... 5-54 Text Telephone (TTY) Users ............................... 7-6 Theft-Deterrent Feature .................................. 3-140 Theft-Deterrent Systems ................................... 2-17 Content Theft-Deterrent ................................ 2-17 PASS-Key® III+ Electronic Immobilizer ............ 2-19 PASS-Key® III+ Electronic Immobilizer Operation ................................................ 2-19 Throttle, Adjustable .......................................... 2-26 Tilt Wheel ........................................................ 3-8 Time, Setting .................................................. 3-86 Tire Pressure Light ............................................. 3-45 Tires ............................................................. 5-64 Aluminum or Chrome-Plated Wheels, Cleaning ............................................... 5-118 Buying New Tires ........................................ 5-84 Chains ....................................................... 5-90 Changing a Flat Tire .................................... 5-92 14 Tires (cont.) Cleaning ................................................... 5-119 Different Size .............................................. 5-86 Dual Tire Operation ..................................... 5-75 High-Speed Operation .................................. 5-74 If a Tire Goes Flat ....................................... 5-91 Inflation - Tire Pressure ................................ 5-73 Inspection and Rotation ................................ 5-80 Installing the Spare Tire ................................ 5-98 Pressure Monitor Operation ........................... 5-77 Pressure Monitor System .............................. 5-75 Removing the Flat Tire ................................. 5-98 Removing the Spare Tire and Tools ............... 5-93 Secondary Latch System ............................ 5-105 Sidewall Labeling ......................................... 5-66 Spare Tire ................................................ 5-112 Storing a Flat or Spare Tire and Tools .......... 5-108 Terminology and Definitions ........................... 5-70 Uniform Tire Quality Grading ......................... 5-86 Wheel Alignment and Tire Balance ................. 5-88 Wheel Replacement ..................................... 5-88 When It Is Time for New Tires ...................... 5-83 Tow/Haul Mode ............................................... 2-34 Tow/Haul Mode Light ....................................... 3-51 Towing Recreational Vehicle ..................................... 4-45 Towing a Trailer .......................................... 4-50 Trailer Recommendations .............................. 4-81 Your Vehicle ............................................... 4-45 Traction StabiliTrak® System ....................................... 4-6 Trailer Towing Recommendations ............................. 4-81 Transmission Fluid, Automatic .................................. 5-21, 5-24 Transmission Operation, Automatic .................... 2-28 Transportation Program, Courtesy ...................... 7-10 Trip Odometer ................................................ 3-35 Truck-Camper Loading Information .................... 4-42 Turn and Lane-Change Signals .......................... 3-9 Turn Signal/Multifunction Lever ........................... 3-9 U Ultrasonic Rear Parking Assist (URPA) ............... 2-62 Uniform Tire Quality Grading ............................ 5-86 Universal Home Remote System ....................... 2-74 Operation ................................................... 2-75 V Vehicle Control ........................................................ 4-4 Loading ...................................................... 4-32 Running While Parked .................................. 2-56 Symbols ......................................................... iv 15 Vehicle Customization, DIC .............................. 3-76 Vehicle Data Recording and Privacy .................. 7-16 Vehicle Data Recording, Radio Frequency (RFID) ........................................ 7-18 Vehicle Identification Number (VIN) ............................................ 5-121 Service Parts Identification Label .................. 5-121 Vehicle, Remote Start ....................................... 2-7 Ventilation Adjustment ...................................... 3-33 Visors ........................................................... 2-17 Voltmeter Gage .............................................. 3-41 W Warning Lights, Gages, and Indicators ............... 3-33 Warnings DIC Warnings and Messages ........................ 3-66 Hazard Warning Flashers ............................... 3-8 Safety and Symbols ......................................... iv Wheels Alignment and Tire Balance .......................... 5-88 Different Size .............................................. 5-86 Replacement ............................................... 5-88 When It Is Time for New Tires .......................... 5-83 Where to Put the Child Restraint ....................... 1-48 Windows ....................................................... 2-14 Manual ...................................................... 2-15 Power ........................................................ 2-15 Sliding Rear ................................................ 2-16 Windshield RainsenseTM II Wipers .................................. 3-11 Washer ...................................................... 3-12 Washer Fluid .............................................. 5-39 Wiper Blade Replacement ............................. 5-64 Wiper Blades, Cleaning .............................. 5-118 Wiper Fuses ............................................. 5-122 Wipers ....................................................... 3-10 Winter Driving ................................................ 4-28 X XM Radio Messages ..................................... 3-117 XMTM Satellite Radio Antenna System .............. 3-142 Y Your Vehicle and the Environment ....................... 6-2 16	https://manuals.plus/m/947f2189054e0269d0d3ea65684f9816460d28fcd988d473204642457fbe3a64_doc
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Industry Profile: Jim Hagerman of Hagerman Audio Labs \| Stereophile.com Industry Profile: Jim Hagerman of Hagerman Audio Labs <here is a image 4e92885a7ec917a6-9bc74d7b44b73e52> This week's Industry Profile features Jim Hagerman of Hagerman Audio Labs. In essence, Jim is Hagerman Audio Labs all-in-one, as he impressively functions as owner/designer/engineer/tester/builder/shipper/customer service, and so on. Hagerman Audio Labs first caught my eye a couple years back when I started getting into DIY projects. Though he has since eliminated DIY from his offerings (you can read why below), Jim continued to interest me because: 1) his website lists an address in Honolulu (a rarity in high-end audio), 2) he publishes his schematics, and 3) his candid approach to social media is refreshing—all hopeful indicators that he'd make a very interesting Industry Profile subject. Additionally, his Trumpet Reference—MM/MC vacuum-tube phono preamplifier, has been a StereophileClass A Recommended Component for a few years now. During a trip back home to O'ahu for the holidays, I reached out to Jim and was kindly invited to visit his home, which doubly functions as his live-in audio workshop. I started off by asking him to share more about his background in life, audio, and electronics. Jim Hagerman:I grew up in Minnesota and went to college there. There weren't any jobs at the time so I went east to Boston. A friend of mine had gone over there and said I should check it out. For a couple years I did that, didn't think I'd ever leave. I grew up in the Midwest and had no desire to go crazy traveling. I spent eight years in Boston, and that's where I met my ex-wife. We went to San Diego after that. It was good for me career-wise. I worked at Digital Equipment, so I worked on all kinds of different types of equipment for a long time. In SD, I was at Hughes Aircraft and Nokia; worked on mobiles phones, TVs. At Hughes, we invented what they call the "D-ILA", now owned by JVC. They're large-screen TV projectors. (The technology was actually invented by Hughes about 30 years ago.) We were doing the first movie-theater-sized electronic projection, at least in color. There was also General Electric, Eidophor, and some others that were doing things. That was fun. Analog electronics. For me, it helps in the audio world, especially doing radio frequency stuff. When you're up in the megahertz and gigahertz, it's a different world. Very analog. Everything—position, trace length, RF, digital, it's all very critical. That's what I bring to the audio world that I think a lot of other people don't have. <here is a image b76982c954f10201-5cf0b908031544e6> Jana Dagdagan:Were you always an audiophile? JH:Well, I've got the same story as everyone else who got in this business. You're 16, you go into an audio store, and you're just blown away by something. For me, it was Klipsch horns, a tube amp, and a Supertramp album: Crime of the Century. We were just stunned. The guy brought us in (knowing he wasn't going to sell us anything), sat us down, turned it way up, and left the room. It was just breathtaking. I ended up buying my first audio equipment from those guys. (This was in Minneapolis.) I got the Advent Model 300, which was kind of a cult receiver at the time, and little Advent speakers. Since then, I grew up playing trumpet, so I knew how to read music and play. I was in the jazz band and orchestra . . . but I kind of sucked! (Laughs) I wasn't a "musician type" and . . . I'll confess I'm not really an audiophile, like most of our customers. JD:If you're not an audiophile, then what are you? JH:I'm a designer. An inventor/designer/creative type. I'm not a musician, and I'm not really an audiophile. I don't collect. I do it mostly as a business. I do it to try and understand what the customer likes and dislikes. But technicallyI'm not an audiophile myself. My advantage is: I'm a designer—a product designer and circuit designer, so I was able to bring my skills into this arena. JD:But...you must be an audiophile to somedegree to be in this industry, right? JH:Actually, in the early '90s, I was going to do guitars, guitar amplification. The guy I was working with, Nestorovic—his Dad, Mile, used to work for Mcintosh and then he started his own company. He had a speaker design . . . He mentioned that his father wanted to get out of the business and sell it. I thought about it but decided to go with guitars instead because it was a bigger market. Vacuum tubes were a big business, and everybody who knows how to design with them is either retiring or dead. There's nobody left. It's just a couple of young guys trying to pick up the torch. There were a handful of people doing it way back when. I thought, "Oh, that's it! I'm going to learn these things." I really got into it. I read everything and studied for years. I figured there was a business in there for me somewhere in the vacuum tube world. I thought it was going to be guitar amps and preamps, maybe pedals. But somehow, I don't know how I got diverted. I decided to do a phonostage, because that's the most difficult thing you can do, right? The high-powered amp is one end, the line stage in the middle is the easiest, and phonostages are probably the hardest. Tiny, tiny signals, you're fighting noise, hum, and everything else . . . After that, I wrote a paper on RIAA networks for an audio publication in '95. So I thought, "okay I'm going to try high-end audio!" And I did. <here is a image 2dedc5d4beef662d-ad49ca98890f4db5> JD:Is Hagerman Audio Labs just you? JH:Yes, it's just me. I build everything from scratch myself. I always thought it would grow into something larger, but for that I'd need a partner. I need the marketing and sales side; my business is completely missing that half. JD:You used to make DIY kits for your affordable components, right? Are you not doing DIY anymore? JH:Everything I used to do was a kit. I've taken them all off the site. It started as a good business, but you don't make any money that way, and people kind of look down on it. They think, "Oh, it's a kit, there are compromises, it's not as good as it could be, you have to sacrifice things to make it a kit, it's not a quality product." No. These are some really good things. Here I'm giving you world-class technology and sound. So yeah, I quit doing kits. A lot of people were just cheap and wanted to save 50 bucks. There were all these people with no skills building kits. I'd be getting calls/emails all the time. People would send me broken machines to fix and send back. I spent way more time on the kits than anything else. It wasn't actually profitable, so I slowly killed them all off. I'm trying to get rid of that "oh, it must be low quality because it's a kit" stigma. It was a good way to get into the business from the bottom up. You could offer a board for 30 bucks and people would love it. You'd kind of build a little bit of a cult following. I don't visit the bulletin boards or forums anymore, but people used to pass the word on and say, "I built this thing and it works great!" So it didhelp build the business, but at this point it's just not worth the trouble for me. JD:What are your thoughts on digital? JH:I gave up digital in 2008. I refuse to touch that stuff any more. The digital world pissed me off so much with the DRM. I had a really good DAC that would do Red Book CDs but then it got crazy with all these different formats and data rates. A universal product is really compromised because you've got too much going on in one box. JD:Any opinions on MQA? It doesn't make you want to revisit digital? JH:No opinion other than it looks like a really clever technology. I don't know how it sounds but it's a clever way to combine everything and be compatible. I've heard some horrible stories about MQA and licensing. They're brutal with their licensing for manufacturers who want to use it and employ it. They sting you. For the small guy like me, I don't think I could afford to get into that business. So nope! Not going back. I've been an analog guy all my life. Digital seemedlike the right thing to do so I brought a very analog approach to when I designed that [the DAC], but I don't want to get into that world anymore. I'd rather just buy vinyl. <here is a image bdcc130f5d75b89d-8ad9d881ff6f1dc8> JD:What's in your listening system? JH:Mostly my own equipment. Right now I'm using a heavily modified Rega turntable with a VTA adjuster and a new counterweight on the tonearm. Other than that all the electronics are my own. Whatever I happen to have on hand, I'll use for a phono stage. The step-ups are from Bob's Devices. Bob Sattin makes a killer product—the best thing for the dollar. And it's [my system's] just headphones right now, I don't have room for speakers. JD:What are you listening to right now? JH:Dave Brubeck's Time Out, Michael Hedges' Aerial Boundaries, Patricia Barbara ( Nightclubhas a couple of tracks on it that are just fantastic for listening deep, deep down into the noise floor, where you're listening to decays. I spend a lot of time listening to the quiet passages, because you need a really good signal to noise ratio, because you've got all this hiss that's playing at the same time. The music level can go well below the level of hiss. And that doesn't happen with digital; the decay just ends and is gone.) JD:What do you love/not love about this industry? JH:What I don't love: The hype marketing. Like "put this crystal in the other room", "put a chocolate chip on top of your tonearm and everything will sound better butit's a $300 chocolate chip". It's a lot of bullshit and they don't back it up with anything but complete gobbledygook. There's no science or physics behind it. There're a lot of people with integrity and then there're the snake oil guys who're just trying to get rich. I don't like the snake oil. I'm the only guy publishing schematics online. Like, here's what you're buying, this is the real stuff. And . . . there is a downside. I do get copied. There's this guy in Hong Kong doing an exact copy of my Bugle, shipping it for half the cost. What I love: the people. I totally love the shows, hanging out; it's a great group of people who love what they do. I wish I was more participatory and could go to more shows, but it's expensive. Especially with living out here. JD:Is there anything you wish audiophiles would understand better about designing? JH:The key is topology. It's not just the parts you use. That's where I come in—I can invent new topologies and improvements. A lot of guys take an existing design and try to improve on it by using better and better parts. But you could take your basic phono stage and you could throw a million dollars of parts at it, and it'd only go so far. You can throw all the money in the world at it, but a better topology with cheap parts would completely toast it. JD:Who do you respect, in terms of design? JH:1) Alan Blumlein: one of the greatest inventors of all time. He invented stereo—but that's not his good stuff; the good stuff is the differential pair, the resonant sweep circuit for horizontal on television (the most brilliant circuit ever!) 2) Tesla, 3) Thomas Edison. And two of the modern day: Jim Williams and Bob Pease. Super gifted electrical engineers who could think outside the box.	https://www.stereophile.com/comment/563059
Refworld \| U.S. Committee for Refugees and Immigrants World Refugee Survey 2007 - India Refworld is the leading source of information necessary for taking quality decisions on refugee status. Refworld contains a vast collection of reports relating to situations in countries of origin, policy documents and positions, and documents relating to international and national legal frameworks. The information has been carefully selected and compiled from UNHCR's global network of field offices, Governments, international, regional and non-governmental organizations, academic institutions and judicial bodies. U.S. Committee for Refugees and Immigrants World Refugee Survey 2007 - India Publisher United States Committee for Refugees and Immigrants Publication Date 11 July 2007 Cite as United States Committee for Refugees and Immigrants, U.S. Committee for Refugees and Immigrants World Refugee Survey 2007 - India , 11 July 2007, available at: https://www.refworld.org/docid/469638821e.html [accessed 4 May 2023] Disclaimer This is not a UNHCR publication. UNHCR is not responsible for, nor does it necessarily endorse, its content. Any views expressed are solely those of the author or publisher and do not necessarily reflect those of UNHCR, the United Nations or its Member States. Refoulement /Physical Protection India reportedly deported a number of Myanmarese from Mizoram State, but the Office of the UN High Commissioner for Refugees (UNHCR) had no access to them and was not able to determine if they were seeking asylum. Indian police also attempted to turn a Myanmarese asylum seeker over to the Myanmarese embassy in New Delhi, but officials there refused to accept him. India also deported a number of Bhutanese refugees to Nepal, where the Government confined them to camps after they tried to cross the Indo-Bhutanese border. India was not party to the 1951 Convention relating to the Status of Refugees and had no refugee law but, in 1996, its Supreme Court ruled that the 1950 Constitution's guarantees of life and personal liberty protected refugees from refoulement. India's Citizenship Amendment Act of 2003 defined all non-citizens who entered without visas as illegal migrants, with no exception for refugees or asylum seekers. India treated refugees differently depending on their nationality. It generally granted Tibetans and Sri Lankan Tamils protection. Nepalis could enter India freely and those with documentation enjoyed most of the rights of Indian citizens under the 1950 Peace and Friendship treaty between the two countries. Other refugees had to travel to New Delhi to reach UNHCR, which had no presence at the borders, and which some refugees reported to be neither fair nor prompt in its status determinations. India requested that UNHCR not register any Bhutanese asylum seekers as refugees, because a 1949 agreement between India and Bhutan (updated in February 2007) granted them legal residence and most rights on par with Indian nationals. Their Government, however, had expelled them and would not issue documents attesting to their Bhutanese citizenship. Nearly 16,500 refugees arrived in Tamil Nadu from Sri Lanka during the year. By the end of 2006, UNHCR had recognized about 11,600 refugees under its mandate. It recognized just over 1,200 asylum seekers during the year, granting refugee status to 550. More than 600 had pending claims at year's end. During 2006, 35 to 45 asylum seekers arrived in New Delhi monthly. The Government ordinarily issued residence permits to pre-2001 Afghan refugees and Myanmarese refugees recognized by UNHCR; other refugees generally did not receive official government documentation authorizing their residence in India. Registration with UNHCR took about seven months to a year to complete. India also hosted a number of Bangladeshi Hindus who Islamic extremists evicted from their land in Bangladesh and generally allowed them to live freely in West Bengal and Tripura States, although it considered them economic migrants. About 35,000 Chakma and Hajong refugees from Bangladesh lived in Arunachal Pradesh, awaiting Indian citizenship. Despite a 1996 Supreme Court decision in favor of this, the State Government resisted. About 70 individuals with refugee status voluntarily returned to their country of origin with UNHCR's help. About 200 refugees resettled to third countries. Detention/Access to Courts India detained at least four refugees in West Bengal State on immigration charges, holding them past the completion of their trials. It released one of them, an Iraqi, to UNHCR, and two others from Myanmar obtained court orders directing their release to UNHCR. The Government detained five refugees and two asylum seekers in New Delhi on immigration charges and one refugee on other criminal charges. In December, a Chin asylum seeker whose claim UNHCR had denied launched a hunger strike outside UNHCR's New Delhi office. UNHCR complained to the Indian police, who arrested him and attempted to turn him over to the Myanmarese embassy. When the embassy would not accept him, the police held him on immigration charges that could ultimately lead to five years in prison. India continued to operate two closed camps for Sri Lankans it alleged to be members of the Liberation Tigers of Tamil Eelam (LTTE) and their families but allowed no judicial oversight of these administrative decisions. India transferred at least 12 new arrivals there in 2006, two for suspected LTTE ties, and 10 for allegedly smuggling asylum seekers across the Palk Strait between Sri Lanka and India. The Constitution reserved to citizens its protections against state discrimination on the bases of race, religion, place of birth, and other grounds, but extended to all persons its right to equality before the law and the equal protection of the law, protection of life and liberty, and prohibition of unlawful detention. The Foreigners Act contained broad powers of detention and made illegal entry into the country a crime punishable by up to five years in prison, with no exception for refugee or asylum seekers. The National Human Rights Commission was able to monitor detention facilities, but did not have a mandate specifically to protect refugees and asylum seekers. In New Delhi, UNHCR provided legal aid through an implementing partner. The Government gave residence permits to many Afghans and Myanmarese. In Mizoram, Myanmarese Chin the police caught without documentation had to pay bribes of 200 to 500 rupees ($4.50 to $11) to avoid deportation. India issued identity documents to Tibetans and Sri Lankan Tamils that legalized their stay in the country. UNHCR provided the refugees it recognized with papers, but India did not officially recognize these. UNHCR also provided documentation to asylum seekers, which authorities generally respected. Freedom of Movement/Residence India maintained around 100 camps for destitute Sri Lankan refugees in Tamil Nadu. Residents were free to live outside them if they chose but had to return every two weeks for attendance and to receive aid. Those who lived in camps but worked outside had to return daily by 7 p.m. India required Sri Lankans and other recognized refugees living outside the camps to register with the nearest Foreigners Regional Registration Office, usually in the local police station. They had to remain in the district unless they received permission, usually granted for economic or other reasons, to move to another. Refugees in New Delhi and Faridabad regularly reported harassment by officials when they tried to change their registered residences, but UNHCR generally intervened to gain permission for them. Refugees with UNHCR papers only had difficulty leaving New Delhi. India did not permit Myanmarese refugees registered with UNHCR to live in its northeastern provinces, near the Myanmar border. In order to rent apartments, Chin refugees in Mizoram State had to obtain letters from the local government and the Young Mizo Association (YMA), a local nationalist group with strong influence over the local government. The YMA illegally deported many Chin in 2003 when the Government considered issuing them work permits. Its members conducted inspections to make sure Chin had the required recommendation letters and deported those it found without them. The Constitution reserved to citizens its rights of freedom of movement and choice of residence. The Foreigners Act and the 1948 Foreigners Order implementing it gave the Government the power to force all foreigners, including refugees and asylum seekers, to "reside in a particular place" and "[impose] any restrictions on [their] movements" and to prosecute criminally anyone aiding or abetting their escape. India rarely issued international travel documents to long-term Tibetan refugees. The Foreigners Order also prohibited refugees and asylum seekers from leaving India without permission. At the end of 2006, India banned Tibetans who entered the country with valid visas and subsequently applied for residence permits from receiving international travel documents. Right to Earn a Livelihood Though there was no legal basis for doing so, India generally allowed Tibetan and Sri Lankan refugees to work. Sri Lankan refugees living in camps often had to turn over their Government aid to corrupt officials to leave the camp for work. Tibetans could obtain certification to practice professions, but other groups of refugees could not. Refugees recognized by UNHCR had no access to legal employment, but often worked in the informal sector. Many worked as street vendors, a highly visible job which made it easy for police to extort money or goods from them. Employers often underpaid them, and landlords overcharged them because they knew the refugees could not report them. The Constitution reserved to citizens its rights to work, practice professions, join unions, and operate businesses. As foreigners, refugees could not legally own land, but Tibetan refugees often acquired land with Indians acting as proxies. Refugees without documentation, including Nepalis, could not open back accounts. Public Relief and Education Much of the housing in Sri Lankan camps was of poor quality, but the Government gave residents subsidized rice and other goods. Government hospitals provided free medical services near the refugee camps. India's provision of public relief and assistance to Tamil refugees approached that of nationals. India gave no aid to Bangladeshi and other refugees whom it did not recognize. Refugees had to wait nearly two months for UNHCR reimbursement for medical visits, and this was generally not available for private hospital visits. All refugees with Government permission to stay in the country had full access to local schools. UNHCR provided language training to assist children in making the transition to Indian schools. Children of families without Government permits had to rely on reimbursement from UNHCR for school fees. Children of Sri Lankan Tamil refugees in Tamil Nadu attended primary school free of charge and received one daily meal, free textbooks, bus passes, and school uniforms. Parents received financial assistance if they lived in one of the refugee camps. Nepalis with documentation could freely attend public schools, but those without had to pay for private schools. India did not allow UNHCR or NGOs to enter Mizoram State, but, in May, a new State Government in Tamil Nadu granted both national and international agencies access to refugee camps. The Foreigners Act authorized the central Government to restrict access to aliens it designated to particular areas.	https://www.refworld.org/publisher,USCRI,,,469638821e,0.html
Entropy \| Free Full-Text \| Information Equation of State Landauer’s principle is applied to information in the universe. Once stars began forming there was a constant information energy density as the increasing proportion of matter at high stellar temperatures exactly compensated for the expanding universe. The information equation of state was close to the dark energy value, w = -1, for a wide range of redshifts, 10 > z > 0.8, over one half of cosmic time. A reasonable universe information bit content of only 1087 bits is sufficient for information energy to account for all dark energy. A time varying equation of state with a direct link between dark energy and matter, and linked to star formation in particular, is clearly relevant to the cosmic coincidence problem. In answering the ‘Why now?’ question we wonder ‘What next?’ as we expect the information equation of state to tend towards w = 0 in the future.c Information Equation of State by M. Paul Gough Space Science Centre, University of Sussex, Brighton, BN1 9QT, United Kingdom Entropy 2008 , 10 (3), 150-159; https://doi.org/10.3390/entropy-e10030150 Abstract Landauer’s principle is applied to information in the universe. Once stars began forming there was a constant information energy density as the increasing proportion of matter at high stellar temperatures exactly compensated for the expanding universe. The information equation of state was close to the dark energy value, w = -1, for a wide range of redshifts, 10> z >0.8, over one half of cosmic time. A reasonable universe information bit content of only 10 87 bits is sufficient for information energy to account for all dark energy. A time varying equation of state with a direct link between dark energy and matter, and linked to star formation in particular, is clearly relevant to the cosmic coincidence problem. In answering the ‘Why now?’ question we wonder ‘What next?’ as we expect the information equation of state to tend towards w = 0 in the future. Keywords: Information ; Energy ; Landauer’s principle ; Cosmology PACS codes: 89.70.Cf, 95.36.+x 1. Introduction Landauer showed that any erasure of information is necessarily accompanied by heat dissipation [ 1 ]. When a bit in the memory of a computer is forced to a standard state, usually by being reset to zero, irrespective of its present value, which may not be known, the information bearing degrees of freedom and thus the entropy of the computer memory are clearly reduced. A corresponding minimum k B T ln2 of heat energy has to be dissipated into the surrounding environment to increase the environment’s thermodynamic entropy in compensation, and in accord with the second law of thermodynamics. The total amount of information is conserved as the surrounding environment effectively contains the erased information, although clearly no longer in a form that the computer can use. Landauer’s principle was originally proposed to describe the energy dissipation when information is overwritten in computer systems and subsequently used to predict the future limits to shrinking computer circuit size [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Shannon information entropy of a computer memory device, log 2 N L , is just the number of bits needed to distinguish between the N L information bearing degrees of freedom used for computer logic states [ 10 ]. In comparison, thermodynamic entropy, k B lnN accounts for the N discrete Boltzmann microscopic states of all constituent atoms, electrons, etc. In equivalent units, the thermodynamic entropy of such a computer memory device is therefore many orders of magnitude larger than the information entropy. The energy dissipated by present day computer memory through the Landauer’s principle is thus miniscule in comparison to normal thermal dissipation. However information entropy is equivalent to thermodynamic entropy when the same degrees of freedom are considered. The information entropy of the physical world is thus the number of bits needed to account for all possible microscopic states. Then each bit of information is equivalent to ΔS = k B ln2 of thermodynamic entropy leading to Landauer’s principle that ΔS T = k B T ln2 of heat is dissipated when a computer logic bit is erased. 2. Applying Landauer’s principle to the universe In the computer memory example of our introduction information ‘erasure’ was shown to be a misnomer as the information present in the computer logic is not destroyed on memory reset but just converted into a form that computer logic can no longer access. Henceforth we enclose the term ‘erasure’ in quotes to represent this action. Each bit of logic ‘erased’ is converted into k B T ln2 of heat energy with an associated increase in the states of the environment surrounding the memory device and overall information is conserved. More generally, Landauer’s principle applies to all systems in nature so that any system, temperature T, in which information is ‘erased’ by some physical process will output k B T ln2 of heat energy per bit ‘erased’ with a corresponding increase in the information of the environment surrounding that system. Landauer’s principle is fully compatible with the laws of thermodynamics [ 9 , 11 , 12 , 13 , 14 ]. Indeed Landauer’s principle has been used to reconcile the operation of Maxwell’s Demon with the second law of thermodynamics. The ‘erasure’ of information when the Demon resets its one bit of memory in preparation for the next measurement is the source of heat dissipation that ensures that there is no overall reduction in entropy [ 3 , 9 , 11 ]. Landauer’s principle can be derived from microscopic considerations [ 13 ] as well as derived from the well-established properties of the Shannon-Gibbs-Boltzmann entropy [ 14 ]. The principle thus appears to be fundamental and universal in application. Landauer’s principle applies both to classical and to quantum information. A foundational principle of quantum mechanics has been proposed [ 15 ] that all elementary systems carry one bit of information. When N initially separate and independent elementary systems interact the assemblage will still carry N bits of information, even if there is complete entanglement, provided there is no information exchange with the environment [ 15 ]. Recently Landauer’s principle has been applied to quantum information theories of vacuum entanglement, holographic dark energy, and black hole physics [ 16 , 17 ]. For example, dark energy may arise from quantum information loss at the cosmic horizon [ 17 ]. Information in the universe is continuously operated on and changed by physical processes [ 18 ]. It is estimated that the universe contains some 10 90 bits and that around 10 120 bits have been processed to present [ 18 , 19 , 20 ]. Note that 10 90 bits is significantly less than the theoretical maximum universe information content of 10 123 bits provided by applying the holographic principle [ 18 , 21 ] to the universe’s surface area. In the maximum information scenario, corresponding to the universe being one single black hole, 10 123 elemental squares of Planck length size are needed to cover the surface of the present known universe. In the regular operation of computers there are many occurrences of data overwriting and resetting with associated heat dissipation from information ‘erasure’ the norm and only avoided by utilising special reversible logic circuits or procedures [ 1 , 2 , 4 , 5 , 8 ]. Thus it seems reasonable to assume that some natural physical processes may also lead to similar heat dissipation from ‘erasure’ of some of the 10 90 bits of information intrinsic to the physical world. Fortunately, for the purpose of deriving this information energy contribution to the universe energy balance it is not necessary to quantify what fraction of the information processed is being, or has been, ‘erased’ in these processes, or even to identify the possible physical processes responsible for ‘erasure’. It is sufficient to be able to say information may be ‘erased’ and thus allow us to equate each bit to k B T log2 of energy that can be dissipated as heat. Such equivalence is standard in cosmology and not fundamentally different from the way we use mc 2 to represent the contribution of all mass to the total universe energy budget when to date only a small fraction of baryon mass has been converted into fusion energy within stars. Most importantly note the equivalent energy of a bit depends solely on the temperature of the system of microstates described. This work takes an information based approach concentrating on just the recent epoch when stars were formed, in contrast to other works which consider the more difficult accounting of information entropy over the whole time span since the big bang [ 18 , 19 , 20 , 22 , 23 ]. 3. Information equation of state: just prior to star formation The universe temperature for the period of time between decoupling and the commencement of any significant star formation is given approximately by the temperature, T , of a radiating universe with the same energy density as our mass dominated universe, with total mass density, ρ tot (including dark matter): ρ tot c 2 = σ v T 4 (1) The (volume) radiation constant, σ v , is defined in terms of the Stefan-Boltzmann (area) constant, σ a , by σ v = 4σ a /c . Replacing σ a by its definition in terms of fundamental constants and applying Landauer’s principle we obtain equation (2), the energy, E , associated with each bit: E = k B T ln2 = (15 ρ tot h 3 c 5 / π 2 ) ¼ ln2 (2) Equation (2) shows that the characteristic bit energy, E , was proportional to ρ ¼ , and thus to a -¾ , where a is the universe scale size. We expect from the second law that total information did not decrease and so we can assume that the evolution of the universe total information bit content lay between two limits. At one limit the total bit content remained fixed with the information bit number density falling in parallel with the density of matter as a -3 . At the other limit the number of microstates increased with increasing volume to provide a constant bit number density. These simply defined limits have been chosen to be broad enough to include previously considered cases [ 18 , 19 , 20 , 22 , 23 ] and at the same time still provide a clear comparison against the period of star formation discussed next. Thus information energy density varied with universe scale size as a -3.75 if total information bit content remained constant, and as a -0.75 if information bit density was constant. These variations (as a -3(1+w) where w is the equation of state) correspond to an information equation of state, w i , just prior to star formation bounded by the range: -0.75< w i < +0.25. 4. Information equation of state: during star formation Landauer’s principle clearly identifies temperature as the single parameter relating information to energy. As stars formed an increasing fraction of the universe bits represent information associated with the very high temperatures of stellar interiors, >10 7 K. Note that the relative difference between the interior temperatures of stars of different types and at different nuclear burning stages, or ages, is small compared with the many orders of magnitude these stellar interior temperatures exceed the temperature of non-stellar matter, primarily in the form of gas and dust. Accordingly, during the early rapid stellar evolution the average temperature and hence average characteristic information bit energy changed primarily in proportion to the fraction of all bits accounting for information in stars. As this information accounts for the Boltzmann microstates of matter, we expect that fraction equals the fraction of all baryons that are in stars. The upper plot in Figure 1 reproduces results from the recent (2006) survey of measurements of high redshift galaxy populations by Hu and Cowie [ 24 ]. The fraction of all baryons, f b , that are in stars has been replotted as a function of universe scale size, a , (replotted from figure 1 of [ 24 ]). To simplify modelling the data has been split into two regions by the vertical line: the main region of rapid change with redshifts, z >0.8; and a more constant region z <0.8. Data points for z >0.8 are reasonably well described by a power law variation with the best fit for f b (solid line) corresponding to a +3.6 and the best fit for the specific variation as a +3.0 is also drawn (dashed line) for comparison. A detailed fit to the data might entail at least three separate periods of fit with the steepest period in the middle, but, for data z>0.8, it is sufficient for our purposes to show that f b varies close to a 3 . The lower plot in Figure 1 illustrates how this increasing fraction of baryons in stars affected the average baryon (and bit) temperature. The majority of baryons, non stellar gas and dust, continued to cool as a -0.75 reaching a temperature today only one order of magnitude higher than the 2.7K temperature of the cosmic microwave background radiation, CMB, consistent with the present matter energy density four orders of magnitude higher than the CMB energy density (see equation 1). However, the proportion of baryons in stars increased as a +3.0 reaching a maximum of around 10% at z=0.8 , causing the average bit temperature to also increase as a +3.0 reaching a temperature of a few times 10 6 K (10% of typical stellar temperatures) at z=0.8. An average bit temperature rising as a +3.0 is the specific characteristic needed to counteract the a -3.0 bit density dilution caused by the increasing separation between formed stars as the universe expands. Thus, for the period 10>z>0.8 , there was a near constant overall information energy density ( a 0 ), or an information equation of state, w i ~ -1.0. In recent times z<0.8 the data points vary less and the fraction of baryons in stars has reached a maximum, ~10 -1 , necessarily limited by definition to f b <1. For reference, the black square in Figure 1 corresponds to the present day estimated fraction of all baryons that are in galaxies [ 24 , 25 ]. Figure 1. Upper plot . The cumulative fraction of all baryons, f b , that have formed stars plotted as a function of universe scale size, a , from Hu & Cowie [ 24 ]. The black line is the best power law fit for data z >0.8 (vertical line limit) and the dashed line the best a +3.0 fit. The reader is referred to [ 24 ] for details of all experimental techniques and corrections applied in this survey of measurements. Lower plot . The increasing fraction of baryons found in stars during the period 10> z>0.8 causes the overall average baryon (and bit) temperature to also increase as a +3.0 . Figure 1. Upper plot . The cumulative fraction of all baryons, f b , that have formed stars plotted as a function of universe scale size, a , from Hu & Cowie [ 24 ]. The black line is the best power law fit for data z >0.8 (vertical line limit) and the dashed line the best a +3.0 fit. The reader is referred to [ 24 ] for details of all experimental techniques and corrections applied in this survey of measurements. Lower plot . The increasing fraction of baryons found in stars during the period 10> z>0.8 causes the overall average baryon (and bit) temperature to also increase as a +3.0 . 5. Overall time history of w i The overall time history of the information equation of state is summarised in Figure 2 . Before star formation commenced the combination of expansion and cooling lead to an information equation of state lying between the limits: -0.75< w i < +0.25. During stellar formation the proportion of high temperature bits increased close to a +3 counteracting the a -3 dilution effects from increasing star separations as the universe expanded. This resulted in a nearly constant overall information energy density, or w i ~ -1.0, for the major part of cosmic time, 10> z >0.8. During this rapid growth in stars, the subsequent changes in stellar temperature with star type and age after birth would have had little effect on the average bit temperature compared with the large temperature increase at star formation. For recent times, z< 0.8, the previous rapid growth in f b slowed to reach a limit with the fraction of all baryons that are in stars ~10 -1 today as star birth and death rates become similar. Now the distribution of star types and temperature evolution with age becomes important. A detailed account of these effects to estimate the variation in average bit temperature in this recent period is beyond the scope of this work. We can assume that w i has remained significantly negative, at least initially, after z= 0.8 as the average temperature continued to increase, primarily determined by the stars formed around z~ 0.8 as they move through hydrogen burning to hotter helium and later burning stages. In the future the overall distribution of stellar temperatures is expected to change less, leading to a more constant average temperature and a tendency for w i ➔ 0. Figure 2. Time variation of the information equation of state, w i . Figure 2. Time variation of the information equation of state, w i . 6. Discussion We have shown that the information equation of state was negative and equal to the dark energy value w DE ~ -1.0 during the rapid star formation period 10> z >0.8, or for over one half of cosmic time. Any negative equation of state, and specifically the value w i ~ -1.0, implies that information energy must make a contribution to dark energy. The universe information energy total is given by the product of total universe information content with the average information bit energy. The lower plot of Figure 1 shows that the average bit temperature since z= 0.8 is around 2x10 6 K (~10% of typical stellar temperatures) and thus the average bit energy around 120eV, from k B T ln2 . Then, in order for the information energy to account for all dark energy, the total universe information content would need to be ~10 87 bits. This value is not unreasonable and is similar to the previous estimates of 10 90 bits [ 18 , 20 ]. However, care must be taken to avoid a circular argument and not to rely solely on this similarity to support our argument since the 10 90 bits value of the previous work [ 18 , 19 , 20 ] was effectively estimated by dividing the universe total mass energy by the characteristic energy of our equation (2). Any significant information energy contribution to dark energy is directly relevant to the cosmic coincidence problem as it implies a natural coupling between dark energy and matter. In particular, the information component of dark energy identified here is directly related to the degree of stellar evolution, consistent with dark energy only affecting the universe since sometime after stars began forming. In providing some response to the cosmic coincidence ‘Why now?’ question we are lead to the important ‘What next?’ question. A full calculation of the average bit temperature after z=0.8 requires a sum over all star types taking into account their different temperature histories and will be the subject of subsequent work. Nevertheless, we can expect w i ➔ 0 in the future since the extent of stellar formation has reached a maximum. Whilst we expect dark energy to remain the dominant energy component of the universe, dark energy density will no longer remain constant but will eventually fall along with mass energy density as a -3 , or w i =0. The universe will continue to expand but no longer with an accelerating expansion. In common with previous work [ 18 , 19 , 20 ] we have concentrated on information associated with baryons, ignoring the information energy density contributions from other universe components such as dark matter or the cosmic microwave background, CMB. Other components may have similar information bit contents but their information energy densities will not be significant as characteristic temperatures are orders of magnitude below stellar temperatures. Dark matter is thought to be present at a mass density ~5x the mass density of ordinary matter. Most explanations for dark matter involve either cold dark matter models or primarily cold models involving a mixture of mostly cold with some hot dark matter. In any case, the average dark matter temperature is thought to be <<10 7 K. CMB can be considered a ‘snapshot’ of the early universe, effectively possessing an information content equal to that of the early universe, and therefore similar in value to today’s universe, assuming CMB and the universe have both conserved information content in the intervening period. Then the very low 2.7K CMB temperature means the CMB information energy density is also insignificant compared with the hot stellar baryon component. In the above estimation of w i during stellar evolution we have ignored the loss of entropy, or reduction of information, resulting from the reduction in the number of microstates assumed to take place on star formation as initially highly disordered matter collapses to form the more ordered structure of a star [ 26 ]. However, this reduction in bit number is small compared with the major increase in temperature at star formation and will have no effect on our calculated equation of state parameter. We can illustrate this by assuming the information contained by any physical system must be similar to the information required to fully simulate that system on a computer. For example, a full simulation of baryons during star formation would require a resolution at least of the order of the Planck length, 1.6 x 10 -35 m. Consider changing from describing a baryon location within the universe (~10 26 m) to a location within a typical star, for example the sun (~10 9 m). This corresponds to a change in accuracy from one part in 6x10 60 (~2 202 ) to one part in 6 x 10 43 (~2 145 ). In order to maintain accuracy sufficient for a full simulation the minimum number of bits required to represent that location parameter on the computer only changes from 202 bits to 145 bits. Such a small 28% reduction in information is miniscule compared with a typical five orders of magnitude increase in temperature at star formation. The reduction in thermodynamic entropy on star formation is usually made compatible with the second law and the ‘arrow of time’ by assuming there is at least a compensating increase in gravitational entropy [ 26 ]. Alternatively, Landauer’s principle leads us to expect that the reduction of entropy, or information ‘erasure’ from reduction of Boltzmann microstates, on star formation dissipates heat to raise the entropy of the surrounding stellar environment in compensation and in accord with the second law of thermodynamics and conservation of information. It has been noted before [ 27 ] that equation (2) above for the characteristic energy, E , of an information bit in the universe, excluding star formation, is identical in form and value to the characteristic energy of a cosmological constant, one of the possible descriptions for dark energy. Taking ln2 ~1 , equation (2) is identical to equation 17:14 of reference [ 28 ]. With the present mass density equation (1) gives an average gas and dust temperature of 35K, and equation (2) provides the 3x10 -3 eV bit energy or characteristic energy that has been associated [ 28 ] with a cosmological constant. Thus our information based argument naturally explains this low value as k B T ln2 – a value previously thought ‘difficult to explain as it is too small to relate to any interesting particle physics’ [ 28 ]. Finally, Figure 1 clearly shows that the earlier rapid a 3 rise in the fraction of baryons in stars ceased around z= 0.8. Although those stars formed at that time will continue to evolve through higher temperature nuclear cycles the average bit temperature clearly no longer rises as steeply as a 3 . We therefore expect to see some observable change to the dark energy equation of state towards a value w> -1 in the most recent times. The contribution information energy makes to dark energy might therefore be verified experimentally by one of the future experiments planned to measure the effects of dark energy with greater precision. 7. Summary By considering a simple model for the equation of state of information in the universe we have shown that information energy must make a significant contribution to dark energy. While the approach taken here may be considered unconventional, the conclusion is supported by a number of strong arguments: The information equation of state was clearly negative for at least one half of cosmic time, 10> z >0.8, with a value close to the dark energy w ~ -1.0. Information energy can easily account for all of the dark energy with an information bit content of ~10 87 bits, similar in magnitude to previous estimates for the universe bit content. The equation for the characteristic bit energy of non-stellar information is identical in form to the equation for the characteristic energy associated with a cosmological constant. The low characteristic energy associated with dark energy was previously thought difficult to explain from particle considerations but is a natural result of this information approach. Information energy is directly related to the degree of stellar evolution, and thus can provide an answer to the fundamental cosmic coincidence question ‘Why now?’ Occam’s razor argues strongly for such a simple explanation to the main (70%) energy component of the universe. 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[ Google Scholar ] © 2008 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). MDPI and ACS Style Gough, M.P. Information Equation of State. Entropy 2008, 10, 150-159. https://doi.org/10.3390/entropy-e10030150 AMA Style Gough MP. Information Equation of State. Entropy. 2008; 10(3):150-159. https://doi.org/10.3390/entropy-e10030150 Chicago/Turabian Style Gough, M. Paul. 2008. "Information Equation of State" Entropy10, no. 3: 150-159. https://doi.org/10.3390/entropy-e10030150 Article Metrics	https://www.mdpi.com/1099-4300/10/3/150/html
Applied Sciences \| Free Full-Text \| Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant In this paper, we are discussing a research project aiming to optimize the scheduling of production orders within a real application in the packaging field. As a first approach, we model the problem as an extended version of the hybrid and flexible flowshop scheduling problem with precedence constraints, parallel machines, and sequence-dependent setups. The optimization objective considered is the minimization of the total tardiness. To tackle this problem, we use two methodologies: mixed-integer linear programming (MILP) and constraint programming (CP). These two models were further extended by adding resource calendar constraints named also availability constraints; this implies that the tasks should be scheduled only when the machine is available. The different proposed models were compared to each other on a set of generated benchmarks that reflect the specific properties of the industrial partner. Finally, as the studied configuration relies on practical real-world application, where thousands of orders are produced monthly, a novel dedicated heuristic was designed to address the need for quick solutions. The latter outperforms the other proposed algorithms for expected total tardiness minimization. The proposed problem can be readily modified to suit a wide range of real-world situations involving the scheduling of activities that share similar characteristics. Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant by Soukaina Oujana 1,2,* , Lionel Amodeo 2,* , Farouk Yalaoui 2 and David Brodart 1 Brodart SAS, 1 Rue du Stand, 10700 Arcis-sur-Aube, France Authors to whom correspondence should be addressed. Appl. Sci. 2023 , 13 (10), 6003; https://doi.org/10.3390/app13106003 Received: 14 April 2023 / Revised: 10 May 2023 / Accepted: 10 May 2023 / Published: 13 May 2023 (This article belongs to the Special Issue Design and Optimization of Manufacturing Systems ) Abstract In this paper, we are discussing a research project aiming to optimize the scheduling of production orders within a real application in the packaging field. As a first approach, we model the problem as an extended version of the hybrid and flexible flowshop scheduling problem with precedence constraints, parallel machines, and sequence-dependent setups. The optimization objective considered is the minimization of the total tardiness. To tackle this problem, we use two methodologies: mixed-integer linear programming (MILP) and constraint programming (CP). These two models were further extended by adding resource calendar constraints named also availability constraints; this implies that the tasks should be scheduled only when the machine is available. The different proposed models were compared to each other on a set of generated benchmarks that reflect the specific properties of the industrial partner. Finally, as the studied configuration relies on practical real-world application, where thousands of orders are produced monthly, a novel dedicated heuristic was designed to address the need for quick solutions. The latter outperforms the other proposed algorithms for expected total tardiness minimization. The proposed problem can be readily modified to suit a wide range of real-world situations involving the scheduling of activities that share similar characteristics. Keywords: scheduling ; optimization ; mixed-integer linear programming ; constraint programming ; dedicated heuristic ; tardiness 1. Introduction Effective production planning and scheduling attract continuous interest from manufacturing companies, which is a good way to add flexibility to the business, to meet the deadlines promised to the customer, and to ensure the best production efficiency by balancing production needs with available resources, all at minimal cost. From this point of view, the use of robust tools for production scheduling remains a strategic issue because they enable optimizing production and meeting market challenges. Scheduling is the operational organization of production in the workshop by deciding the order in which tasks pass through the machines, respecting a certain number of constraints to which the workshop is subjected, and according to optimization criteria considered for decision making. In other words, the schedule can be defined as follows: assign the task ‘ i ’ to the machine ‘ k ’ at a given time ‘ t ’ while considering, for example, the operator ‘ p ’ equipped with the tool ‘ o ’ and the mater ‘ m ’. Among different workshop configurations, a flowshop scheduling problem (FS) arises in the context of repeated production, where jobs are required to visit the stages in the same order and undergo identical processing operations; in other words, all operations of all tasks go through the machines in the same order. In order to cope with real-world problems, improve the overall capacity, add additional flexibility to the production, and avoid bottlenecks if some operations are too long, it is possible to multiply the number of machines that can perform the same operation. The resulting model is known in the literature as hybrid flowshop (HFS), also called flowshop with parallel machines; it consists of a set of processing stages, in which each stage may have several identical or non-identical machines, with at least one stage having two or more parallel machines. The classical hybrid flowshop assumes that all jobs need to visit all stages in the same order. However, in practice, each job might miss out or skip some stages, which can improve the performance of the model and make it better suited for real-world industrial settings. HFS scheduling problem with stage skipping is also called hybrid flexible flowshop scheduling problem. The configuration under study is described in Figure 1 . Figure 1. Studied configuration. In hybrid and flexible flowshop (HFFS) scheduling problems, two decisions should be taken: the assignment of jobs to the parallel machines as well as the sequencing of the jobs allocated to each machine. This problem is known to be NPhard in its simple version and in most of its extensions. As an example, Hoogever et al. [ 1 ] demonstrated that preemptive scheduling in a two-stage flowshop with at least two identical parallel machines in one of the stages so as to minimize makespan is NP-hard in the strong sense. Gupta et al. [ 2 ] considered a non-preemptive two-stage hybrid flowshop problem in which the first stage contains several identical machines and the second stage contains a single machine; they demonstrated that the problem is NP-hard in the strong sense even when there are only two machines at the first stage. HFFS scheduling problem has been widely applied in various manufacturing environments, and several realistic constraints were considered. A fair amount of research has focused on a variety of realistic constraints, ranging from sequence-dependent setup times, constraint calendar, transportation time, due dates, and so on. Furthermore, several optimization criteria were considered, covering the commonly used makespan, costs, transportation, maximum tardiness and earliness, and the total of tardy job. In this paper, we make three significant contributions. Firstly, we introduce novel CP and MILP models that take into account specific constraints, including sequence-dependent setups and resource calendar constraints. Secondly, we assess the performance of both models using real industrial benchmarks. Lastly, we propose a dedicated heuristic that effectively addresses the need for fast computation times in practical real-world applications, such as the one studied in this paper, where thousands of orders are produced each month. The remainder of this paper is organized as follows: Section 2 reviews the state of the art regarding the related papers. The problem description is presented in Section 3 . In Section 4 , we formulate all the proposed resolution models (MILP, CP, and a novel dedicated heuristic). The computational experiments, allowing to evaluate the performance of the proposed models, are presented in Section 5 . Finally, in Section 6 , we present the conclusions of our work. 2. State of the Art The area of flowshop scheduling has been a very active field of research. It was first proposed by Johnson in 1954. Since then, several approaches have been proposed and numerous optimization objectives were considered. The current trends that attracted researchers during the last decade in scheduling problems are toward integrating practical constraints. Among all, we can point out setup time, resource calendar, and machine flexibility. 2.1. Constraints 2.1.1. Setup Constraints Setup time, also called changeover, is a very important factor in the packaging industry because it may have a significant impact on the overall production cycle. It denotes the required time interval to prepare the necessary material resources. In many real-life situations, a setup often occurs while shifting from one operation to another. Setup time is classified into two categories: sequence-independent setup time and sequence-dependent setup time. Sequence-independent setup time depends solely on the current task regardless of its previous task. Sequence-dependent setup time depends on both the current and immediately preceding task [ 3 , 4 ]. There has been a growth in interest in incorporating setup times in many studies. The main reason why researchers have been motivated to utilize this assumption is to solve scheduling problems in a real manner [ 5 ] Liu and Chang [ 6 ] addressed the problem of F m \| S t s d , C s d , r i \| ∑ S T i , C i . They first formulated the problem as an integer programming problem. Then, they employed a Lagrangian relaxation approach and finally developed a search heuristic. Three major types of heuristics were proposed by Kurz and Askin [ 7 ], who explored the F m \| S t s d , C s d , r i \| ∑ C i problem, namely insertion heuristics (based on insertion heuristics for the traveling salesman problem), Johnson’s algorithm, and a set of naïve greedy heuristics. They investigated these three patterns and identified the range of conditions under which each method performs well. Salmasi et al. [ 8 ] proposed a mathematical programming model for F m \| f m l s , S t s d \| ∑ C i as the problem is proven to be strongly NP-hard; two heuristic algorithms, tabu search (TS) and hybrid ant colony optimization (HACO), were developed to solve the problem. In addition, a lower bounding method based on the branch and price algorithm was developed to assess the performance of the metaheuristic algorithms. An et al. [ 9 ] considered the F 2 \| w t , S t s d \| C m a x problem; they developed several dominance properties, lower bounds, and heuristic algorithms and used the latter to develop an efficient branch and bound algorithm. Cheng et al. [ 10 ] tackled the F p \| S t s d \| C m a x problem; they proposed a mixed-integer linear programming model to solve small-sized instances. Due to the strong NP hardness of the research problem, an effective metaheuristic, called pairwise iterated greedy (PIG) algorithm, was proposed to solve medium- and large-sized problems. Rossi and Nagano [ 11 ] proposed a mixed-integer linear programming (MILP) model for F m \| S t s d \| ∑ T i problem. They proposed a method to evaluate the total tardiness of a permutation sequence and also introduced a partial acceleration method to calculate the total tardiness in an insertion neighborhood. In addition, they developed a new heuristic to solve the problem efficiently. This heuristic was then integrated into the best metaheuristics available in the literature. Kare and Agrawal [ 12 ] studied the F m S t s d ∑ w T i , w E i problem. Three evolutionary metaheuristics were proposed. 2.1.2. Resource Calendar Constraints Another common and practical constraint found in real environments is to consider the resource calendar. The traditional scheduling problem assumes that machines are continuously available. However, in reality, this is often not the case due to non-availability periods, such as maintenance, vacations, leaves, and so on. Considering these time-off periods for resources is crucial for accurate and realistic scheduling. This helps to determine when resources are available to work on assigned tasks, ensuring that work is only scheduled during available times. Machine availability constraints encountered in real-world environments can be classified as either fixed or non-fixed [ 13 ]. For fixed constraints, the intervals of unavailability are predetermined, whereas they are unknown for the non-fixed constraints. Unavailability periods can also be categorized based on operation preemption as non-preemptive [ 14 ], crossable or non-crossable [ 15 ], or resumable, semi-resumable, or non-resumable [ 16 ]. An operation is known as non-preemptive when its processing on a machine cannot be interrupted until it is totally completed, and after that the concerning machine switches to another operation. An operation interrupted by an unavailability period is called resumable when its processing can continue during the next availability period. It is called non-resumable if it has to restart from the beginning when the performing machine is available again. An operation is known as semi-resumable if it has to partially restart during the next available period. There is other terminology introduced by Mauguière et al. [ 15 ]. It concerns unavailability periods allowing interruption of operations: crossable and non-crossable unavailability periods. An unavailability period that allows an operation to be interrupted and resumed after the unavailability period is called crossable, while an unavailability period that does not allow the interruption of any operation is known as non-crossable. Figure 2 gives a description for the notation used for interruptible and non-interruptible operations. Figure 2. Notations for interruptible/non-interruptible operations. Bentalleb et al. [ 17 ] consider a deterministic case where unavailability periods are known in advance and fixed and correspond to preventive maintenance tasks. They tackled a two-machine job shop scheduling problem with an availability constraint on one machine under makespan minimization. First, two mixed-integer programming (MIP) models were proposed and then some heuristics were performed to solve the problem. Azem et al. [ 18 ] investigate the job shop problem where operations can be interrupted by resource unavailability periods. They propose approximation methods based on construction heuristics. Surprisingly, the literature on the flowshop scheduling problems with resource calendar or fixed machine availability is not abundant. Aggoune et al. [ 14 ] address the flowshop scheduling problem with limited machine availability under the makespan criterion and under the assumption that the machines are not available during the whole planning horizon. They propose a heuristic approach based on the geometric approach to approximately solve the problem. Figealska [ 19 ] studied the problem of preemptive scheduling in a two-stage flowshop with parallel unrelated machines under makespan minimization. Heuristic algorithms were proposed based on combined linear programming procedures and a genetic algorithm. Laribi et al. [ 20 ] investigate an extension of the classical flowshop scheduling problem to the case where jobs processing requires additional nonrenewable resources; the goal is to minimize the makespan. They propose an efficient mathematical model. 2.1.3. Machine Flexibility Constraints In a modern manufacturing unit, machine flexibility is a very important feature that enables increasing the overall workshop capacities, reducing or eliminating the impact of bottleneck stages and balancing the capacities of the stages for the overall workshop. Such a production unit is characterized by several stages. Each stage is made up of a set of parallel machines. At some stages, the machines are duplicated and a job can be processed on any machine. A flowshop with parallel machines is also known as a multiprocessor flowshop, flexible flowshop, or hybrid flowshop. Machine flexibility has attracted much attention from researchers in recent years. There are several examples provided in the literature, including steelmaking [ 21 , 22 ], industry [ 23 ], as well as the semiconductor industry [ 24 , 25 ]. Odugawa et al. [ 26 ] provide a survey on several real-world applications, ranging from the metal forming industry to the paper industry to the chemical industry. Some researchers address real-world problems in their papers. Kochhar et al. [ 27 ] exhibit a local search approach to solve highly realistic flexible flow line scheduling with setups, buffer capacities, as well as blocking and breakdowns. Several heuristics are provided by Botta-Genoulaz [ 28 ] for the flowshop scheduling problem with multiple identical machines per stage, precedence constraints and time lags, and setups. Ruiz and Maroto [ 29 ] provide a metaheuristic, in the form of a genetic algorithm, to a complex generalized flowshop scheduling problem that results from the addition of unrelated parallel machines at each stage, sequence-dependent setup times, as well as machine eligibility. Naderi et al. [ 30 ] investigate the problem of hybrid flexible flowshop (HFFS) with sequence-dependent setups, where the objective is to minimize the makespan. They put forward two advanced algorithms that effectively handle the flexible and setup features of this problem. Chen [ 31 ] proposed an integer hybrid metaheuristic based on the principles of variable-neighborhood descent and TS for unrelated parallel machines problems with ready times and sequence- and machine-dependent setup times to minimize the weighted number of tardy jobs. While many papers in the literature have tackled various realistic considerations and constraints, to the best of our knowledge, there has been no effort to jointly address the set of realistic constraints incorporated in the problem formulation of our paper, which include sequence-dependent setups, machine flexibility, and resource calendar constraints. 2.2. Optimization Criteria Setting the correct optimization criteria or objectives for a scheduling problem is not always an easy task as they are diverse, convoluted, and often conflicting. Plenty of scheduling problems have been studied considering several criteria. The most considered are makespan ( C m a x ), total flow time, total tardiness, maximum tardiness, and number of tardy jobs. Makespan and total flow time seek the effective utilization of the manufacturing resources by reducing the elapsed time between the start and the completion of a sequence of operations in a set of machines, while the remaining criteria are related to job due dates. In fact, makespan minimization is significantly important in order to upsurge the utilization of the production system. However, in today’s competitive environment, focusing on makespan minimization without meeting the due date is of no use for an industry since meeting customer deadlines is crucial. According to Sen and Gupta [ 32 ], when a task is not completed before its due date, some penalties are incurred, such as potential loss of customers, damaged reputation, loss of market competitiveness, penalty clauses if there are any, as well as expediting (the job is assigned quickly to the processing machine at the possible cost of extra setups, double handling of material, inefficient use of workmen and machine), etc. Hence, scheduling problems with tardiness objectives have attracted increasing attention from managers and researchers. Table 1 provides a summary of several significant studies that focus on the tardiness objective. The “Constraints” column contains the various constraints that were taken into account in these studies. The “//m” column refers to parallel machines, the “ S T s d ” column pertains to sequence-dependent setups, the “ d i ” column represents due date constraints, the “ w i ” column represents waiting time, and finally the “ r i ” column represents release date constraints. Table 1. Important studies on scheduling problems with tardiness objectives. 3. Problem Description The problem under study corresponds to a real industrial problem of a packaging company that prints, on average, 1000 jobs per month. The operations can be categorized into four main groups, ranging from the preparation of printing materials to the printing process and shifting process (winding, perforation, and coating) and finally shaping process aiming to make orders into their final form. Moreover, it is important to point out that this process is characterized by flexibility, where machines might be skipped and not all the machines must be visited by all the jobs. A job i consists of a number n of operations; each operation O i j can be processed by a subset of machines and has a processing time on machine k , and it may be zero for some jobs as the jobs are not processed in some stages (skipping). Note that p i denotes the processing time of job i . Before starting processing, a setup time (ST) is needed between each of two consecutive scheduled jobs on each machine. That is to say that, to transition from the processing of the current operation O i j to the next one O i ’ j ’ on machine k , some setup settings must be implemented according to the characteristics of each operation, such as color, size, etc. An example of setup for the printing phase consists of removing the ink colors not required for the next job on that printing machine to free up the ink trays for colors that are required for the next job. Transitioning from one job to another requires to change ink colors. The time required to set up one job for the printing phase can be divided into three steps: the first one to empty the tray from the previous ink, the second one to clean the ink tray, and the last one to reload the appropriate ink color. The global needed setup time depends on the number of color changes. On average, the significant setups may contribute to 40% of the global printing step, including processing time and setup time. However, if a job “ i ” requires the same color as the previous job “ i − 1 ” , then the setup time for this color may be avoided because the considered printing machine is already loaded with appropriate color and, therefore, major setups are not needed. The setup time duration is correlated to the setup settings’ similarities between two consecutive operations. The more resemblance the operations’ settings, the shorter the machine setup. Another important feature of the considered problem is resource calendar constraints (RC), which allow to set the work shifts of all machines. The work shift is a segment of continuous available times of a machine. This means that machines are available only during working times in the calendar. These unavailability periods are the consequence of shift patterns or planned maintenance. On the other hand, the machine setup cannot be interrupted by unavailable periods, and the end of the setup must be immediately followed by the beginning of the operation processing. Furthermore, a transportation time is needed to transport a job from the current processing machine to the next one. Based on the key features of the considered production system, a production schedule should be planned to maximize the production effectiveness so that the printing line can gain as much production benefit as possible. The production effectiveness can be represented by an objective function that should be defined based on the production targets of the problem. In flexible manufacturing plants operating in a make-to-order environment, the efficient utilization of manufacturing resources is typically pursued to meet delivery deadlines. Thus, in our case, we aim to minimize the total tardiness of all jobs, meaning that we seek to find a job sequence that minimizes the total amount of time by which all jobs are completed after their due dates. The production problem can be described as a hybrid and flexible flowshop with nineteen unrelated parallel machines, denoted using the classical Graham notation H F F 19 \| P r e c , S T s d , R C , d i \| ∑ j = 1 n t i [ 56 ]. This classification is based on the features mentioned above, and it is commonly used to represent production systems. As the first systematic attempt to solve this problem, we construct a mathematical model in the form of mixed-integer linear program (MILP) that considers sequence-dependent setups; we then add waiting constraints and evaluate how it behaves, and, finally, we added resource calendar constraints that enhanced the complexity of the problem. The objective is to both assign jobs to one machine at each stage and then sequence jobs on machines to minimize the total tardiness. If the completion time of a job is greater than its due date ( C i > d i ) , then it is called tardy and tardiness takes positive values. Otherwise, it becomes an early job with a tardiness value equal to zero ( t i = m a x ( 0 , C i − d i )). 4. Model and Notations Job characteristics are modeled as follows: Let N be the number of jobs to be scheduled. Each job i ( i = 1 … N ) is composed of a set of operations J i that must be processed according the defined processing route. Let M be the number of all available material resources “machines”. For each operation j , let m j ⊂ M be the set of operations that can perform j ∈ j i and p i k be the corresponding processing times. To transition from executing operation O ij to operation O i’j’ on machine k, a setup time s t i j i ’ j ’ k must be incurred. In our problem, the setup time for a job is dependent on the previous job that was processed on the same machine and thus on the job processing sequence. For each machine K k = 1 … m , let l k be the number of unavailability periods and υ k l , υ − k l the time window of unavailability of material resource k ∈ m . The processing of each job on the latter can only be preempted by this interval υ k l , υ − k l and resumed once the machine becomes available. Let d i denote the due date and specify the time limit by which job i ∈ N should be completed. The number of jobs, their respective processing times, and due dates are predetermined and known beforehand. Each machine has a capacity and can only process one job at a time. A machine can only process one operation at a time. The processing of the latter can be interrupted by an unavailability period. Setups cannot be interrupted by an unavailability period and should occur when the machine is available during the setup interval, and, once completed, the processing of the associated operation should start. There is no limit on the capacity of the intermediate stock (buffer) between the production stages. Finally, the objective is to minimize the total tardiness. The notation used in this mathematical modelling is summarized in Table 2 and Table 3 : Table 2. Notation used for the problem data. Table 3. The notation used for the decision variables. 4.1. Mixed-Integer Linear Programming In this section, the MILP formulation presented in [ 3 ] is recalled using the notation of Section 4 and afterwards extended in Section 4.1.2 by adding resource calendar constraints. 4.1.1. Start-Based Model The start-based model was developed in our previous work [ 3 ], with the consideration of sequence-dependent setups, parallel machines, and precedence constraints, and this model is formulated as a mixed-integer linear programming model as below and called M I L P w a v from now on. M i n i m i z e T = ∑ i = 1 n t i (1) Subject to: t i = max ⁡ ( C i − d i ) (2) ∑ k = 1 m e X i j k = 1 , ∀ i ∈ N , j ∈ J i , k ∈ m j (3) C i j k ≥ S i j k + P i j k − B i g M ( 1 − X i j k ) , ∀ i ∈ N , j ∈ J i , k ∈ m j (4) S i j k + C i j k ≤ B i g M ( X i j k ) , ∀ i ∈ N , j ∈ J i , k ∈ m j (5) C i j k ≥ S i j k , ∀ i ∈ N , j ∈ J i , k ∈ m j (6) ∑ i ∈ j ∑ j , j ’ ∈ o i ∑ k ∈ m i Y i j i ’ j ’ k = 1 , ∀ i , i ’ ∈ N , j , j ’ ∈ J i , J i ’ k ∈ m i j ∩ m i ’ j ’ S i j k ≥ C i ’ j ’ k + S t i j i ’ j ’ k − B i g M ( 1 − Y i j i ’ j ’ k ) , ∀ i , i ’ ∈ N , j , j ’ ∈ J i , J i ’ k ∈ m i j ∩ m i ’ j ’ S i ’ j ’ k ≥ C i j k + S t i j i ’ j ’ k − B i g M Y i j i ’ j ’ k , ∀ i , i ’ ∈ N , j , j ’ , J i , J i ’ , k ∈ m i j ∩ m i ’ j ’ C i j k = P i j k + S i j k , ∀ i ∈ N , j ∈ J i , k ∈ m j C i = ∑ k = 1 m i j C i j k , ∀ i ∈ N , j ∈ J i , k ∈ m i j t i ≥ 0 , i ∈ N X i j k ∈ { 0,1 } ; ∀ i ∈ N , j ∈ o i , ∈ m i j Y i j i ’ j ’ k ∈ 0,1 , ∀ i , i ’ ∈ N , j ∈ J i , j ’ ∈ J i ’ , k ∈ m i j ∩ m i ’ j ’ S i j k ≥ 0 , ∀ i ∈ N , j ∈ J i , k ∈ m i j C i j k ≥ 0 , ∀ i ∈ N , j ∈ J i , k ∈ m i j The objective function (1) aims at minimizing the sum of the total tardiness of all jobs. Constraint (2) provide us with the value of the individual tardiness of each job. Constraint (3) states that each operation can only be assigned to one machine, where the decision variable X i j k is non-zero if operation O ij is assigned to processing unit k and zero otherwise. Constraint (4) ensures that a job’s completion time is no earlier than the sum of its start time and processing time. Constraint (5) sets the end date of each job on machines that are not processing the job to 0. Constraint set (6) controls job completion at stages that a job may skip. Constraint set (7) enforces precedence constraints, ensuring that each operation of a job can only begin after its preceding operation has been completed. Constraints (8) and (9) are used together to sequence any pair of tasks ( i , i′ ) assigned to the same processing unit k, preventing two jobs from being processed simultaneously on the same machine to ensure the machine is occupied when processing an operation. Constraint set (10) specifies that the completion time of any operation is the sum of its start time and processing time. Constraint set (11) calculates the completion time of a job as the sum of the completion times of all the operations in its processing route. Constraint (12) ensures that only positive tardiness values are considered. Finally, Constraint sets (13) (14), (15), and (16) define the decision variable domains. 4.1.2. Modeling Calendar Constraints The start-based model is further extended to solve unavailability problems, which are also addressed by incorporating resource calendar constraints. The processing of a job should not start during the time window of unavailability of resource k . That is to say that any operation must be carried out and finished before the arrival of an interval of unavailability. The execution time of an operation must be outside unavailability interval. S i j k , S i j k + P i j k ∩ υ k l , υ − k l = ∅ (17) Constraint (18) allows to calculate the total unavailability period of a machine k that processes job i . u i = ∑ k = m ∑ l l k X i k ( υ − k l − υ k l ) (18) Constraint (19) calculates the operations completion time C i j k = P i j k + S i j k + S t i j i ’ j ’ k + u i , ∀ i ∈ N , j ∈ J i , k ∈ m j (19) Now, we have: C i = ∑ k = 1 m i P i j k + S i j k + S t i j i ’ j ’ k + u i , ∀ i ∈ N (20) From now on, we refer to the model that incorporates resource calendar constraints into model M I L P w a v , M I L P R C . 4.2. Constraint Programming Constraint programming (CP) has good performance and robustness in the optimization field. In fact, it is a strong tool for solving discrete optimization problems; it provides a set of modeling features suitable for a very wide range of complex scheduling problems that do not have a simple formulation. It provides an algebraic language with simple mathematical concepts; commonly, CP framework contains useful structural information; it has the advantage of exposing high declarative, compact, and flexible constraint formulations, which allow us to model the problem correctly and therefore makes it perform well for finding optimal feasible solutions [ 4 ]. Here, we have modeled the problem in CP using IBM ILOG CP Optimizer. We will not provide the details of the modeling language used in this paper. For those interested in learning more about this, we recommend referring to [ 57 ] and the CP Optimizer reference manual. 4.2.1. Start-Based CP Model A formulation of the main variables is presented in Table 4 using the concepts of CP Optimizer. From now on, this model is called C P w a v . Minimize T = ∑ i = 1 n m a x ( 0 , t i ) (21) Table 4. CP decision variables. Subject to: t i = m a x ( 0 , e n d O f ( i t v s [ C i ] ) − d i ) (22) EndBeforeStart ( β j , [ α j k ] ) j ∈ J i , k ∈ m i (23) Alternative ( β j ′ β j ’ ) j , j ′ ∈ J i n o O v e r l a p [ α j k ] j ∈ J , ∀ k ∈ m j (25) d i ≥ β i . e n d ∀ i ∈ N i n t e r v a l α j k , o p t , s i z e = P j k + S t j j ′ k , i ∈ V , k ∈ m i e n d A t S t a r t ( S t j j ′ k , α j k ) j ∈ J , ∀ k ∈ m j The objective function is to minimize the total tardiness (21), given by the difference between the job’s end value and due date (22). The EndBeforeStart constraints (23) represent the precedence constraints between interval variables. Alternative constraints (24) represent the assignment constraints stating that each operation must be performed on exactly one machine. Constraint (25) defines the nonoverlapping constraint; that is to say that, during the interval [ α j k ] , which represents the assignment of an operation j to machine k , the latter cannot overlap; e.g., the machine is busy during this interval. The constraint endAtStart ( α , β ) is used to state that the end of a given interval variable α , equals the start of a given interval variable β . We use this constraint (28) to ensure that the end of a setup should be followed by the execution of the considered operation. 4.2.2. Modeling Calendar Constraints The considered processing line is periodically submitted to calendar constraints; this means that machines are not available during the whole planning horizon. To consider machines’ unavailability, variable α j k should be modulated by adding an intensity step function F k that represents the unavailability interval of machine k. In CP optimizer, Intensity is a stepwise function that applies a measure of usage or utility over an interval length. The intensity is 0% during the unavailability interval υ k l , υ − k l and 100% outside this interval. Therefore, modelling machines’ unavailability can simply be formulated by constraint (29) i n t e r v a l α j k , o p t , s i z e = P j k , i n t e s i t y = F k , j ∈ J , ∀ k ∈ m j (29) An additional feature of our problem is that the setup cannot occur during an unavailability period. To model this feature, we use the predefined constraint forbidExtent (a,U). This expression prevents an interval variable from being scheduled during any time point within the augmented horizon that is not also within one of the disjoint time windows. f o r b i d E x t e n t S t i i ’ k , F k , i , i ’ ∈ N , ∀ k ∈ m i (30) Forbidden start constraint f o r b i d S t a r t α , F states that, whenever the interval is present, it cannot start at a value t wher e F t = 0 . In the same sense, Forbidden end constraint f o r b i d E n d α , F states that, whenever the interval is present, it cannot end at a value t where F t − 1 = 0 . We use constraints (31) and (2) to ensure the respect of unavailability periods. f o r b i d S t a r t α j k , F k , j ∈ J , ∀ k ∈ m i (31) f o r b i d E n d α j k , F k , j ∈ J , ∀ k ∈ m i From now on, we refer to the model that incorporates resource calendar constraints into model C P w a v , C P R C . 4.3. Dedicated Heuristic In order to meet the needs of an industrial environment, we need to be able to develop quick and effective solutions that can be used to solve the various tasks that are involved for a real industrial framework. Unfortunately, the exact resolution approaches presented previously cannot sufficiently address the requirements for real industrial-size instances (more than 100 tasks). A very common difficulty when trying to solve such large-sized instances with the MILP model is running out of memory. The CP model reaches better solutions in a short time, but, similarly, the solver has some issues regarding the instances’ size. In this section, we propose an effective dedicated heuristic that performs well and finds good-quality solutions within a reasonable amount of time. This heuristic follows the logic of a greedy algorithm, which is a type of problem solving technique that involves making a series of decisions in order to find the best solution. It works by making the best decision at each step without considering the long-term consequences of the decisions. The algorithm works by considering the most immediate benefit of each decision and choosing the one that provides the lowest tardiness. This procedure is repeated until all jobs have been inserted, resulting in a complete candidate solution. The main steps of this dedicated heuristic are given below: Step 1. Find earliest schedule Step 2. Check machine’s busyness Step 3. Setting operation’s schedule This heuristic was coded on python. The detailed procedure of the heuristic is presented in Appendix A . 5. Experimental Results 5.1. Performance of MILP and CP Models In this section, the performance of the proposed models is evaluated. We use ILOG Cplex 12.10 software and CP Optimizer (CPO) for solving the MILP model and the CP model, respectively, using a DELL personal computer equipped with an Intel ® Core™ i5-8250U @ 1.6 1.8 GHz CPU, 8 GB RAM, and Window 10 operating system. This section begins with a description of the numerical instances that were tested. Then, the different results tables are presented and, at the end, comparisons between the different algorithms are made. 5.1.1. Test Instances To validate the proposed approaches in this work, we present in this section a description of the test instances that were used. Most of the datasets were initialized on the real database of the studied printing company over a period of 2 weeks. We collected from the production database all the data related to products: operations, processing times, setup times, waiting times, and resource calendar. To test the performance behavior of the proposed solution approaches and to investigate their efficiency, it is necessary to build several sets of instances in various production environments and different conditions. To this end, some test problems have been applied in a variety of conditions with inspiration from the illustrated case study. Each test set is generated by varying the problem size. It can be characterized by N , the number of jobs, O , the total number of operations, and M , the number of machines. The result tables will not mention the number of machines as it remains constant at 19. The different instances are named WOS for Workshop Scheduling followed by the number of the instance. An extensive set of numerical experiments have been conducted by considering different problem sizes. The aim is to investigate which jobs and operations the model is not able to find solutions for in a reasonable resolution time. Based on the combination of the two abovementioned factors, two categories of instances are arranged as the small- and large-sized instances. These categories correspond to different workload situations, respectively: low-workload situation and normal- to high-workload situation. 5.1.2. Experimental Results In this section, we intend to evaluate the proposed models. We set the stopping criteria parameters as follows: the time limit CPU is equal to 30 min and the maximum iterations equal to 1000. The performances of the models are evaluated thanks to real data of the workshop. Test results are discussed below. Several set instance sets have been created with N ranging from - {5, 8, 10, 13, 15 to 20} for small-sized instances. - {30, 40, 50, 65, 70, 75, 80 to 100} for large-sized instances For each set, at least two test instances were generated by varying the number of operations. For each problem class, an effort measurement is completed by calculating the associated total tardiness and the required CPU time. The optimal values that are obtained for tardiness have been distinguished with bold numbers. When applying both formulations to the test instances, a total of 160 experiments were carried out. Instances without Resource Calendar Constraints We now present some results on the solution quality obtained with the different models that do not take into account resource calendar constraints. Small-Sized Instances In this subsection, the general performance of the MILP and CP models is evaluated by a set of small-sized instances. Several instance sets have been created with n ranging from 5 to 20. Table 5 provides an overview of the obtained results. For each problem, the name, the number of jobs, the number of operations and machines, the total tardiness T in minutes, and the solution time in seconds are shown for both models ( M I L P w a v and C P w a v ). Table 5. Main characteristics of the considered small-sized instances and comparison of M I L P w a v and C P w a v models. As the results show, the MILP model provides a great performance; it is capable of solving to optimality all the small-sized problems up to n = 20 and o = 64 within a reasonable time. The CP model, on the other hand, seems to be performing better regarding the resolution time. For all the studied instances, the MILP model took longer to achieve an optimal solution. Figure 3 provides a time comparison between the resolution of the CP and MILP models. We can clearly see that the resolution time difference becomes more noticeable as the number of jobs increases. Figure 3. Resolution time (CPU) comparison between CP and MILP models. 2. Large-Sized Problems To further validate the performance of the proposed models, larger-sized instances are evaluated. Table 6 summarizes the corresponding computational results. Table 6. Main characteristics of the considered large-sized instances and comparison of M I L P w a v and C P w a v models. As can be observed, up to N = 40 and O = 88 , the MIP model is unable to find a solution within 1800 s, whereas the CP model still finds an optimal solution for all instances in 38 s on average. The first conclusion that can be drawn is that CP is much faster than MILP. This experimentation confirms CP’s outstanding performance for the problem under study. Instances with Resource Calendar Constraints This subsection shows the results of the instances on the models that incorporate resource calendar constraints. Small-Sized Instances The results of the computational comparison for each combination of n and m are presented in Table 7 . Table 7. Main characteristics of the considered small-sized instances and comparison of M I L P R C and C P R C models. If we analyze the results when solving the M I L P R C and C P R C models with small-sized instances that consider resource calendar constraints, the presence of a high number of unavailability periods decreases, even more regarding the performance of the MILP model (the model only obtains 15 out of 20 optimal solutions and 20 of 20 feasible solutions). The CP model, on the other hand, seems to perform well and is still able to obtain optimal solutions even when the number of unavailabilities is high. 2. Medium- and Large-Sized Instances The computational results for the medium/large-sized problems are summarized in the Table 8 below. Table 8. Main characteristics of the considered large-sized instances and comparison of M I L P R C and C P R C model. The experimental results show that the M I L P R C model is not able to solve any problem of size up to 30 jobs, while the C P R C model solved a large number of instances up to 75 jobs. 5.1.3. Discussion According to the results of the experiments, the CP algorithm is more efficient than the MILP model when it comes to solving our scheduling problem for both w a v (without ressource calendar constraints) and R C formulations (with resource calendar constraints). It can perform well in handling any size of problem and proves the optimality of a large number of instances. Even with high-availability periods, the CP model can still find optimal solutions. It also proves the optimality of several instances and outperforms the MILP model when it comes to finding feasible solutions. Summing up, we can clearly see that the computational effort required to solve our scheduling problem depends on the size of instances and the number of unavailability periods. The difference between CP and MILP increases as the number of jobs and the number of unavailability periods increase. CP can provide significant savings in computational effort compared to MILP formulation and finds better solutions and is the best overall in all instances. 5.2. Dedicated Heuristic For testing the performance of the proposed dedicated heuristic method, we generated a benchmark composed of several sets of instances with different problem sizes by using the real data obtained from the manufacturing environment of the plant. Accordingly, there are 10 groups of benchmark problems of different sizes, varying from 60 to 150 jobs. Table 9 provides for each instance the tardiness found, denoted by T D h , as well as the execution time (CPUs column) to reach the best value. The column denoted by T r e a l recalls the real results obtained by the planner. Finally, the gap between both solutions is calculated in the column (gap). The last row represents the average values. The values denoted in bold indicate that the heuristic reaches the optimal value for the considered instances, meaning that the solution found by the heuristic is equal to the one obtained by the exact method “CP”. For instances up to 80 tasks, the results obtained with the MILP and CP models are not provided since the solver ran out of memory before providing any initial solution. Table 9. Main characteristics of the considered large-sized instances and comparison of M I L P R C and C P R C models. According to Table 9 , if we compare the results of the dedicated heuristic against the real results obtained by the planner, we see that the tardiness obtained by the heuristic is significantly lower than that obtained by the planner, with an average gap of 39%. On average, the dedicated heuristic provides a better solution overall for all the tested instances within a reasonable time compared to the real solution proposed by the planner, which proves the efficiency of the dedicated heuristic. 6. Conclusions This paper aims to apply operations research techniques to schedule activities within a packaging company. It examines a difficult scheduling problem, which involves a hybrid and flexible flowshop with various challenging features, such as parallel machines, precedence constraints, sequence-dependent setup times, and resource calendar constraints. The paper presents and analyzes two solutions for the problem using MILP and CP Optimizer. MILP is a general-purpose solver, while CP Optimizer is specifically designed for scheduling problems and has its own modeling language. The study compares the effectiveness of the IBM ILOG CPLEX MILP and IBM ILOG CP Optimizer solvers based on their ability to handle realistic problem sizes, with some showing promise on small instances but struggling on larger ones. From the foregoing, MILP formulation performed well for small-sized instances but struggled to find solutions for large-sized instances, or ones with a high proportion of unavailability periods. The CP formulation performed better for large-sized instances and ones with a high proportion of unavailability periods. Therefore, CP Optimizer is more successful in finding optimal solutions for a greater number of instances than MILP. To deal with large-sized instances, a dedicated heuristic was also proposed to provide good-quality solutions in reduced time. Thus, this heuristic is mainly recommended for large-size problems. Future work should focus on improving the proposed algorithm by adding some dispatching rules and investigating a comparable method for resolving scheduling issues with restricted availability, where operations may be suspended due to availability periods and resumed later, with or without incurring penalties. Author Contributions Writing—original draft, S.O.; Writing—review & editing, L.A. and F.Y.; Supervision, D.B. All authors have read and agreed to the published version of the manuscript. Funding Institutional Review Board Statement Informed Consent Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidentiality of the company. Acknowledgments The authors would like to express their gratitude to the editorial board of the journal as well as the anonymous reviewers. Conflicts of Interest The authors declare no conflict of interest. Appendix A Table A1. Pseudo algorithm of the dedicated heuristic. Table A1. Pseudo algorithm of the dedicated heuristic. Pseudo-Algorithm **************** SCHEDULING METHODS **************** Determine an operation’s schedule --------------------------------- a. initialize - l = operation’s processing time (setup+execution) - ls = operation’s setup time - s = start - ss = None, the actual start, es = None, the start of the execution, r = None, the available time - ee = s, the end of the execution - A = machines’ availabilities (list of int couples representing each an availability window) b. iteration - b = 0, the availability bucket - While l>0 and b<\|A\| (we still processing time and availability buckets • B = A[b], B is the current availability bucket • si = B[0] (interval start), ei = B[1] (interval end) • if ei<=ee (if this intervals ends before the moving counter ee) ○ continue to next interval c. Set the availability time, r = ee + operation’s waiting time Find earliest schedule ---------------------- a. Try to schedule at time - determine a timing from time timing = determineTiming() - check if the machine is busy any time between timing.start and timing.end busy = checkBusy() - if not(busy) • return timing and end b. Else, try to schedule at each busyness interval’s end for [si,ei] in the machine’s busyness intervals - if ei<time => skip and continue to next interval - timing = determine a timing from ei - busy = check if machine is busy in that timing - if not(busy) • return timing and end Check machine’s busyness ------------------------ Setting operation’s schedule ---------------------------- a. Set the operation’s attribute (start,exec,end,available,machine) to (timing[1],timing[2],timing[3],timing[4],machine.id) b. Add the interval [timing[1],timing[3]] is the machine’s busyness and reorder the busyness intervals by increasing values c. 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Objective Function Year Author Reference Constraints Approach //m S T s d d i w i r i Total weighted tardiness 1997 Lee and Pinedo [ 33 ] ✓ ✓ ✓ Dispatching rule ATCS (Apparent Tardiness Cost with Setups) 2000 Park et al. [ 34 ] ✓ ✓ Dispatching rule 2009 Naderi et al. [ 35 ] ✓ ✓ MIP and EMA metaheuristic 2013 Xi and Jang [ 36 ] ✓ ✓ Dispatching rules (ATCS) 2020 Diana et al. [ 37 ] ✓ ✓ VND metaheuristic Total tardiness 2009 Chen [ 31 ] ✓ ✓ ✓ Hybrid Approach (ATCS+SA) 2014 Herr and Goel [ 38 ] ✓ ✓ MIP 2015 Liang et al. [ 39 ] ✓ ACO algorithm 2018 Lee [ 40 ] ✓ Random iteration greedy metaheuristic 2020 Rossi and Nagano [ 11 ] ✓ MILP, heuristics and metaheuristics Makespan and total tardiness/tardy jobs 2009 Naderi et al. [ 41 ] ✓ ✓ SA algorithm 2013 Tran et Ng [ 42 ] ✓ A hybrid water flow algorithm 2018 Allahverdi et al. [ 43 ] ✓ AA algorithm 2021 Wan et al. [ 44 ] A pseudo-polynomial algorithm and a dual FPTAS 2022 Allali et al. [ 45 ] ✓ MILP and metaheuristics (GA, ABC, MBO) Tardy jobs 2017 Aydilek et al. [ 46 ] A DR algorithm 2019 Najat et al. [ 47 ] ✓ Mathematical programming and heuristics 2021 Della Croce et al. [ 48 ] ✓ Exponential time approximation algorithms 2022 Hejl et al. [ 49 ] ✓ A decomposed ILP model Bi-objective Sum of weighted earliness and weighted tardiness 2008 Behnamian et al. [ 50 ] ✓ A hybrid metaheuristic algorithm that combines ACO, SA, and VNS 2009 Behnamian et al. [ 51 ] ✓ Three hybrid metaheuristics 2011 Behnamian et Zandieh [ 52 ] ✓ ✓ ✓ A discrete colonial competitive algorithm 2019 Otten et al. [ 53 ] ✓ Heuristic 2020 Schaller and valente [ 54 ] ✓ BB and heuristics 2020 Kellerer et al. [ 55 ] FPTAS Table 2. Notation used for the problem data. Problem Data i , i’ Index for jobs where i , i ’ ∈ {1,…,N}. j Index for operations. O The total number of operations. O ij The j t h operation of job i ∈ N. k Index for machines where k ∈ {1,…, m }. M Number of all material resources. N Number of jobs to be scheduled. J i Set of operations of job i ∈ N. P i Processing time job i ∈ N. d i Due date of job i ∈ N. m j ⊂ M Set of material resources that can perform the operation j ∈ ji . S t i j i ’ j ’ k Setup time to pass from the execution of an operation O j to operation O j ’ on machine k. BigM A very large number. m i j ∩ m i ’ j ’ Set of machines on which operations j of job i and j’ of job i’ can be processed. l k The number of unavailability periods on machine k ∈ m . υ k l The starting time of the l t h unavailability period of material resource k ∈ m . υ − k l The ending time of the l t h unavailability period of material resource k ∈ m Table 3. The notation used for the decision variables. Decision Variables X i j k = 1 if the operation O ij is assigned to the material resource k . 0 otherwise. Y i j i ’ j ’ k = 1 if the operation O ij is processed before the operation O i’j’ on the material resource k . 0 otherwise. S i j k = Starting time of the operation O ij on machine k . C i j k = Completion time of the operation O ij on machine k . C i = Completion time of job i. Table 4. CP decision variables. Decision Variables i n t e r v a l β j = An interval variable for each operation j i n t e r v a l α j k = An optional interval variable for each possible assignment of operation j to machine k ∈ m j Table 5. Main characteristics of the considered small-sized instances and comparison of M I L P w a v and C P w a v models. Instance Characteristics M I L P w a v C P w a v Instance N O T M I L P C P U M I L P T C P C P U C P WOS1 5 7 0 5 0 4 WOS2 5 12 0 8 0 4 WOS3 5 20 0 12 0 4 WOS4 8 10 0 11 0 4 WOS5 8 25 0 50 0 5 WOS6 8 30 0 69 0 5 WOS7 10 17 0 58 0 5 WOS8 10 29 0 110 0 5 WOS9 10 43 0 180 0 12 WOS10 10 49 0 270 0 12 WOS11 13 18 0 90 0 12 WOS12 13 34 0 250 0 21 WOS13 13 49 0 360 0 21 WOS14 15 20 2870 240 2870 21 WOS15 15 45 4076 300 4076 32 WOS16 15 53 5760 410 5760 32 WOS17 15 59 7120 360 7120 32 WOS18 20 29 8200 380 8200 45 WOS19 20 55 9590 520 9590 40 WOS20 20 64 14,200 730 14,200 45 Average 12 33 2591 221 2591 18 Optimal values in bold. Table 6. Main characteristics of the considered large-sized instances and comparison of M I L P w a v and C P w a v models. Instance Characteristics M I L P w a v C P w a v Instance N O T M I L P C P U M I L P T C P C P U C P LWOS1 30 66 0 320 0 6 LWOS2 30 80 5760 850 5760 6 LWOS3 40 88 9852 1710 9712 6 LWOS4 40 96 _ >1800 9980 6 LWOS5 50 110 _ >1800 12,100 12 LWOS6 50 127 _ >1800 19,560 12 LWOS7 65 143 _ >1800 19,800 12 LWOS8 65 150 _ >1800 21,600 30 LWOS9 65 165 _ >1800 22,400 26 LWOS10 65 185 _ >1800 23,980 26 LWOS11 70 164 _ >1800 23,800 30 LWOS12 70 172 _ >1800 25,000 42 LWOS13 70 190 _ >1800 26,960 42 LWOS14 75 182 _ >1800 26,740 73 LWOS15 75 198 _ >1800 27,880 73 LWOS16 75 212 _ >1800 29,660 73 LWOS17 80 210 _ >1800 29,920 49 LWOS18 80 225 _ >1800 38,500 70 LWOS19 100 320 _ >1800 40,940 87 LWOS20 100 380 _ >1800 48,520 87 Average 65 173 _ _ 23,141 38 Optimal values in bold. Table 7. Main characteristics of the considered small-sized instances and comparison of M I L P R C and C P R C models. Instance Characteristics M I L P R C C P R C Instance N O U T M I L P C P U M I L P T C P C P U C P RCWOS1 5 7 1 0 10 0 302 RCWOS2 5 12 1 0 14 0 302 RCWOS3 5 20 2 0 18 0 302 RCWOS4 8 10 2 0 15 0 302 RCWOS5 8 25 2 0 58 0 302 RCWOS6 8 30 3 0 82 0 950 RCWOS7 10 17 3 0 68 0 950 RCWOS8 10 29 3 0 140 0 950 RCWOS9 10 43 3 180 240 180 950 RCWOS10 10 49 4 1330 320 1330 950 RCWOS11 13 18 4 685 40 685 950 RCWOS12 13 34 4 1258 380 1258 950 RCWOS13 13 49 4 2780 450 2780 1100 RCWOS14 15 20 4 4200 490 4200 1100 RCWOS15 15 45 5 7200 820 7200 1100 RCWOS16 15 53 5 8400 1080 8320 1100 RCWOS17 15 59 5 9600 1202 8592 1300 RCWOS18 20 29 5 10,800 1440 9987 1300 RCWOS19 20 55 6 _ >1800 12,600 1300 RCWOS20 20 64 7 _ >1800 18,600 1300 Average 12 33 4 2609 326 3783 888 Optimal values in bold. Table 8. Main characteristics of the considered large-sized instances and comparison of M I L P R C and C P R C model. Instance Characteristics M I L P R C C P R C Instance N O U T M I L P C P U M I L P T C P C P U C P LRCWOS1 30 66 1 _ >1800 0 1300 LRCWOS2 30 80 1 _ >1800 6760 1300 LRCWOS3 40 88 2 _ >1800 9900 1300 LRCWOS4 40 96 2 _ >1800 9998 1300 LRCWOS5 50 110 2 _ >1800 13,100 1300 LRCWOS6 50 127 3 _ >1800 19,760 1300 LRCWOS7 65 143 3 _ >1800 20,100 1487 LRCWOS8 65 150 3 _ >1800 21,900 1487 LRCWOS9 65 165 3 _ >1800 23,254 1487 LRCWOS10 65 185 4 _ >1800 23,978 1487 LRCWOS11 70 164 4 _ >1800 23,900 1487 LRCWOS12 70 172 4 _ >1800 26,020 1487 LRCWOS13 70 190 4 _ >1800 26,990 1580 LRCWOS14 75 182 4 _ >1800 26,840 1580 LRCWOS15 75 198 5 _ >1800 28,520 1580 LRCWOS16 75 212 5 _ >1800 29,760 1580 LRCWOS17 80 210 5 _ >1800 _ >1800 LRCWOS18 80 225 5 _ >1800 _ >1800 LRCWOS19 100 320 6 _ >1800 _ >1800 LRCWOS20 100 380 7 _ >1800 _ >1800 Average 65 173 4 _ _ 19,424 _ Optimal values in bold. Instance Characteristics N O M U T r e a l T D h C P U D H Gap 60 148 19 5 30,120 22,695 4 25% 70 160 19 6 48,215 27,458 4 43% 80 189 19 7 68,743 38,548 6 44% 90 210 19 9 80,471 49,895 8 38% 100 260 19 9 94,875 58,951 8 38% 110 298 19 11 100,458 64,251 8 36% 120 352 19 12 124,524 70,589 12 43% 130 397 19 15 150,427 86,758 12 42% 140 410 19 16 159,751 89,827 12 44% 150 480 19 20 180,058 118,745 19 34% 105 290 19 11 103,764 62,772 9 39% Optimal values in bold. MDPI and ACS Style Oujana, S.; Amodeo, L.; Yalaoui, F.; Brodart, D. Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant. Appl. Sci. 2023, 13, 6003. https://doi.org/10.3390/app13106003 AMA Style Oujana S, Amodeo L, Yalaoui F, Brodart D. Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant. Applied Sciences. 2023; 13(10):6003. https://doi.org/10.3390/app13106003 Chicago/Turabian Style Oujana, Soukaina, Lionel Amodeo, Farouk Yalaoui, and David Brodart. 2023. "Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant" Applied Sciences13, no. 10: 6003. https://doi.org/10.3390/app13106003 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Article Metrics No Article Access Statistics For more information on the journal statistics, click here . Multiple requests from the same IP address are counted as one view. Zoom \| Orient \| As Lines \| As Sticks \| As Cartoon \| As Surface \| Previous Scene \| Next Scene Cite Export citation file: BibTeX \| EndNote \| RIS MDPI and ACS Style Oujana, S.; Amodeo, L.; Yalaoui, F.; Brodart, D. Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant. Appl. Sci. 2023, 13, 6003. https://doi.org/10.3390/app13106003 AMA Style Oujana S, Amodeo L, Yalaoui F, Brodart D. Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant. Applied Sciences. 2023; 13(10):6003. https://doi.org/10.3390/app13106003 Chicago/Turabian Style Oujana, Soukaina, Lionel Amodeo, Farouk Yalaoui, and David Brodart. 2023. "Mixed-Integer Linear Programming, Constraint Programming and a Novel Dedicated Heuristic for Production Scheduling in a Packaging Plant" Applied Sciences13, no. 10: 6003. https://doi.org/10.3390/app13106003 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Appl. Sci. , EISSN 2076-3417, Published by MDPI	https://www.mdpi.com/2076-3417/13/10/6003/htm
Seven Days in Solitary [5/18/20] - Solitary Watch Seven Days in Solitary [5/18/20] Our Weekly Roundup of News and Views on Solitary Confinement by Valerie Kiebala\| May 19, 2020 • The Philadelphia Inquirerpublishedthe “Lockdown Diaries,” a set of writings from incarcerated people during Pennsylvania’s longest ever statewide lockdown. Instituted because of the coronavirus pandemic, the lockdown basically places everyone in the prison system in indefinite solitary confinement in an effort to control contagion (despite the existence of more humane and effectivealternatives). Accompanied by illustrations created by reporter Samantha Melamed, the diary entries describe what it’s like to be locked in a cell for all but 40 minutes a day and how people spend those precious minutes outside of the cell. Thomas Schilk, held at SCI Phoenix said, “Prison is bad all the time, but the smaller the box you’re forced into, the worse it gets. It’s like being locked in a bathroom for weeks.” Robert Pezzecca said, “I had to use the toilet today in front of my cellmate. Guard ordered me to take down the sheet I had hung up to separate our view of each other. It’s not right. My mental health is suffering.” Another man held at SCI Phoenix, Jorge Cintron, said, “It concerns me how quickly it’s forgotten that not long ago five men committed suicide in two months at SCI Graterford.” According to Cintron, three people have recently died at the prison, two of whom had tested positive for COVID-19. Drawing by Samantha Melamid from “Lockdown Diaries” (Philadelphia Inquirer) • The Marshall Projectpublished anarticlewritten by Wesley Williams, currently held in solitary confinement at Upstate Correctional Facility in New York. Wesley described listening to Governor Andrew Cuomo on his radio “in the box” talking about the “pain of isolation” and the real “emotional trauma” people are now experiencing unable to hug someone or be comforted during this time of sheltering-in-place. Wesley, who has been incarcerated for fifteen years, recalled the last time he remembered receiving a hug: when we was eighteen years old, held at Rikers Island and an older incarcerated man comforted him. Since then, he says he’s felt “no human touch beyond an occasional rough pat-down from a correctional officer.” Wesley writes, “No human being deserves to rot away in an 8-by-12 box, in solitary deprivation. Long after COVID-19, there will still be a population here, alone and deprived of human touch.” • The Interceptreportedthat 43-year-old Tiffany Mofield, an incarcerated African American mother of three, died on April 29 in the administrative segregation unit at Edna Mahan Correctional Facility in New Jersey. Mofield had been moved back from the quarantine unit to solitary confinement despite her persisting COVID-19 symptoms. Angelina, another incarcerated woman who witnessed Mofield’s death, said that officers locked Mofield in the shower, though she told officers “she could not breathe.” Angelina said, “She never would have died if she was released from ad-seg and returned to the facilities’ general population. All of us are at heightened risk of death in ad-seg the way it is operated at this facility. Inmates routinely threaten to commit suicide and are routinely ignored.” • Courthouse News Servicereportedon arguments before the Ninth Circuit Court of Appeals, which revisited the terms of a landmark 2015 settlement between the California Department of Corrections and Rehabilitation (CDCR) and a class of 1,600 people held in prolonged solitary confinement in the state’s Security Housing Units (SHU), represented by the Center for Constitutional Rights (CCR). In the settlement, CDCR agreed to end the use of indefinite solitary confinement, which was often based on unconfirmed “gang affiliation.” At the hearing, attorney Samuel Miller argued that men are still being held in solitary confinement, in violation of the agreement. “Half of them are being held in their cells longer than when they were in SHU,” Miller said. While CDCR argued that transferring the men out of the SHU fulfilled the settlement, CCR attorney Rachel Meeropol said, “Being locked in a cell for 23 hours a day is solitary confinement no matter what name the CDCR calls it.” The court has not yet released its ruling. • The Marshall Projectpublished anarticleon the effect that the coronavirus pandemic has had on American juvenile facilities, many of which now hold youth in solitary confinement without rehabilitative or educational programs. Based on lawsuits and interviews with incarcerated youth, the article reports that solitary confinement has “resurged in an effort to socially distance” and now youth are increasingly held in harmful conditions mirroring adult confinement. Amy Fettig, director of the ACLU National Prison Project’s Stop Solitary campaign, said, “As ever, putting kids in solitary, instead of medical isolation monitored and administered by health professionals for the briefest of times needed, is just about administrative convenience for adults.” Data is not available for how many of the 44,000 incarcerated youth across the country are being held in solitary during the pandemic, partially due to facilities barring oversight measures. •Accordingto KATU, seven incarcerated people are filing a lawsuit against Oregon Governor Kate Brown, the Oregon Department of Corrections, and ODOC Director Colette Peters, calling for immediate action to reduce the state’s prison population and increase medical precautions in prisons. The plaintiffs are all over 60 years old and have medical conditions that make them more susceptible to the virus. The state in total incarcerates more than 1,000 people over the age of 60. The director of Civil Rights Project and Oregon Justice Resource Center, Juan Chavez, said that people are being sent to solitary confinement for requesting proper health measures. “People are afraid to speak up or, frankly, be tested if they’re just going to be treated that way,” said Chavez. As of May 12, the ODOC reported 101 incarcerated people and 28 staff had tested positive for COVID-19. • WRALreportedthat community members across North Carolina held a “social distance protest” last week, calling for people to be released from the Central Prison in Raleigh and the Neuse Correctional Institution in Goldsboro. Demonstrators called for an end to the use of solitary confinement and the implementation of proper health care during the pandemic. An organizer of the demonstration, Julie Schneyer, said, “Over years and years, what we’ve seen in DPS [Department of Public Safety] facilities is systematic, ignoring of any kind of human rights…People certainly are extremely concerned, people are terrified behind the wall, they feel like they’ve been given a death sentence.” • The Southern Poverty Law Centerreportedthat a 20-year-old transgender woman faces isolation, transphobic abuse, and inadequate health care at Winn Correctional Center, a facility privately managed by LaSalle Corrections that houses Immigration and Customs Enforcement (ICE) detainees along with Louisiana state prisoners. Karime, an asylum seeker from Mexico, says that she has been sent to solitary “as punishment” for standing near a man and was threatened with solitary upon her request to see a doctor when she became ill. Karime expressed fear for her life in the facility, where she receives physical threats and unwanted sexual advances from men. “I’m depressed and suffer anxiety and insomnia. I almost would’ve preferred being in Mexico, even though I would be harmed more there,” Karime said. “From the moment I wake up until the moment I go to sleep, it’s psychological torture.” • PEN America conducted aninterviewwith Keri Blakinger, a formerly incarcerated reporter for the Marshall Project, about women in prison, the effect of the coronavirus on incarcerated women, and her time in solitary confinement. Blakinger recalled, “I remember going in [to solitary], and I was just blown away by how neon white the walls were, and the window…you can’t actually see out of it…There’s no clock, and there’s nothing. I didn’t have any books…You can hear the muffled screams from the people next to you, but you can’t really hear what they’re saying.” Blakinger continued, “A lot of people say to me that solitary—they like being alone. They’re sure they wouldn’t mind solitary confinement, but solitary confinement is not being alone—it’s more like being buried alive. You lose sense of time. You sometimes question if you still exist, and you don’t know when you’re getting out or when this ends.”	https://solitarywatch.org/2020/05/19/seven-days-in-solitary-51820/?share=twitter
(PDF) QUANTITATIVE AND QUALITATIVE GUT CONTENT ANALYSIS OF MACROBRACHIUM ASSAMENSE PENINSULARE FROM RAWASAN STREAM OF GARHWAL HIMALAYA, UTTARAKHAND, INDIA. PDF \| Food is an essential for any organism, for growth, reproduction and culture aspects. Study on food and feeding of Macrobrachium assamense... \| Find, read and cite all the research you need on ResearchGate PDF Available QUANTITATIVE AND QUALITATIVE GUT CONTENT ANALYSIS OF MACROBRACHIUM ASSAMENSE PENINSULARE FROM RAWASAN STREAM OF GARHWAL HIMALAYA, UTTARAKHAND, INDIA. December 2016 International Journal of Advanced Research 4(12):786-791 DOI: 10.21474/IJAR01/2460 Authors: Hemwati Nandan Bahuguna Garhwal University C.B. Kotnala Govt.p.g.college.rishikesh Abstract Food is an essential for any organism, for growth, reproduction and culture aspects. Study on food and feeding of Macrobrachium assamense peninsulare has been carried out by quantitative and qualitative gut content analysis methods. Prawns were collected monthly from August 2013 to July 2015 and analyses of stomach contact were made to know the natural food of these prawns. From the overall analysis it has been reported that these prawns are omnivorous in nature and generally eat up the microscopic food, either animal or plant matters. Food consists of fragments of aquatic plants, algae, animal matter, diatoms, sand grains and highest percentage of amorphous mass. Results indicate that the adults are mostly predators of benthic invertebrates rather than detritus feeders/scavengers 588a2bb145851594678d53 ae.pdf Content uploaded by Dr-Koshal Kumar Content uploaded by Dr-Koshal Kumar Author content ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 786 Journal Homepage: - www.journalijar.co m Article DOI: 10.21474/IJAR 01/2460 DOI URL: http://dx.doi.o rg/10.21474/IJAR01/2460 RESEARCH ARTICLE QUANTITATIVE AND QUALITATIVE GUT CONTEN T ANALYSIS OF MACRO BRACHIUM ASSAMENSE PEN INSULARE FROM RAWASAN STR EAM OF GARHWAL HIMALA YA, UTTARAKHAND, INDI A. Koshal Kumar 1 , C. B. Kotnala 1 and Anita Rawat Ran a 2 . 1. Department of Zoology, Campus Pauri, H.N. B Garhwal (A Ce ntral University) U ttarakhand India-246001. 2. Department of Zoology, Govt. P.G. (Auto nomous) College Rishikesh, U ttarakhand India-249202. Manuscript Info Abstract Manuscript History Received: 23 October 2016 Final Accepted: 21 November 201 6 Published: Dec ember 2016 Key words:- Macrobrachium assamense peninsulare , natural food, gut c ontent analysi s, Rawasan stream Food is an essential for any organism, for growth, reproduction and culture aspects. Study on food and feeding of Macrobrachium assamense p eninsulare has been c arried o ut by quantitativ e and qualitative gut content analysis methods. Prawns were collected monthly fro m August 2013 to July 2015 and analyses of stomach contact were made to know the natural fo od of these prawns. From the overall analysis it has been reported that these prawns are omnivorous in n ature and generally eat up th e microscopic food, either animal or plant matters. Fo od consists of fragments of a quatic plants, a lgae, animal matter, diatoms, sand grains and highest percentage of amorphous mass. Results indicate that the adults are mostly pr edators of benthic inverteb rates rather than detritus feede rs/scavengers. Copy Right, IJAR, 2016,. All rights reserv ed. Introduction:- Macrobrachium assamense peninsulare, a minor prawn, found in the l ow velocity water bodies in the Uttarakhand, are economically used as food by the local habitants. However, their supply is primarily totally from the wild catch and is limited due t o the inadequate source of availability in their natural ha bitat. The members of gen us Macrobrachium are adapted themselves to a wide range of food of both plant and animal origin. It has been shown from earlier study that these minor prawns are om nivores in their fe eding habits ( Sharma and Subba, 2005 ) . Information about the natural food of the prawn and the ir nutritional characterist ic is essential and act as an evidence for artificial diet during aquaculture. Due to mass culture practices in prawn, the significance of artificial food is recognized. The selection of experimental diet has frequently b een asses sed in term of food conservation ratio ( Froster, 1976; Reddy & Shakuntala, 1986 ). Accurate diet and feeding habits description helps to understand the tropical interaction of aquatic animals with their food web. Feeding is th e most important function of an organism and most of the time in their daily li fe devoted by these organisms in the search of pr oper fo od. Although many authors have studies food and feeding of prawn species earlier (Chopra, 1939; Panikkar, 1952; Sharm a & Subba, 2005; Hall, 1962; Tho mas, 1972; Marte, 1980; Khan, et al., 2001; Bas ker et al., 2013; Bakhtiy ar et al., 2014) This paper deals with th e study of stomach contents of Macrobrachium assamense peninsulare collected from it s natural habitat from R awasan stream to know the natural food co mposition. Corresponding Au thor:- Anita Rawat Rana. Address:- Departme nt of Zoology, Gov t. P.G. (Autonomous) College R ishikesh, Uttarakhand Indi a- 249202. ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 787 Materials and Methods:- To understand the food intake of Macrobrachium assamense peninsulare the qualitative and quantitative analysis of gut contents were made on monthly basis for an interval of two y ears (2013 -2015). For thi s study 10 male an d female were fixed in 70% al cohol soon after their capture, in order to stop the digestion of food in the alimentary canal ( Williams, 1981 ) and brought to the laboratory for further analysis. In the laboratory before processing for the microscopic examination of the food contents, total weight of the individual intact prawn and that of its gut contents were recorded separately . In each specimen the carapace was opened from th e dorsal side of th e body an d gut was removed carefully. A visual examination of fulln ess of intestinal bulb was also made immediately a fter its r emoval (Devi et al., 2013) . The mi croscopic a nalysis was carri ed out as hurriedly as possible and was n ever overdue from more than 4 day af ter capturing the animal. Analysis of Gut con tents:- In p rawn, the food which reaches the gu t is very fine crushed and is meshed due to the presence of p owerful mandible. It is further churned in the mill itself and thus, the food items were identified up to the level which was possible. Different type of foo d i tem and thei r pe rcentage was c alculated on the b asis of fractional analysis, by taking 1 ml of well shaken and thoroughly mixing stomach c ontent suspension from petri dish int o the Sedgewick rafter chamber and examined under binocular microscope to obtain percentage compos ition of di fferent food items. Various food items were examined using binocular microscope and identified to the lowest p ossible t axonomical level with the help of keys an d photographs provided by Ward and Whipple (1959); Needham and Needham (1962) and (APHA, 2012) . The qu antitative analy sis was made and po int was given purely on the basis of observation of fo od items. Observations:- Food and feeding habits:- The mode of feeding in Macrobrachium assamense peninsulare was observed visually in the field during the study. It was observed that prawn, by habit is an omnivo rous species and exhibits feeding activity in day time in comparison to th e other species. These prawns ar e also shy in nature an d hide them self in their m icro habitats and found under the stones, crevices near the bank of the stagnant water during study. Quantitative study:- Monthly visual observation w as also made and reco rded the de gree of fullness (%) of gut in Macrobrach ium assamense peninsulare (Table 2). It is clearly proved that fullness of gastric content is directly related with seasonal variation and 100% fullness o f gastric content was neve r observed during the study period of two years. Quantitative analyses revealed less than 6% population as empty stomach, 59.8% of stomach as actively fed including, 100%, 75% and 50% diet co ntents and poorly fe d having 40.9% of stomach includi ng, trace, empty and 25% gut co ntent. Qualitative stud y:- On the basis of microscopic examination of the gastric c ontent, f ood was identified and categorized into s even groups viz., those co ntaining fragments of aquatic plants, algae, animal matter, diatoms, sand grai ns and unidentified organisms . Different food it ems percentage in male and female pra wn shown in Fig 1 (a & b). Composition of gut contents of Macrobrachium assamense peninsulare in r elatio n t o size groups were pr esented in the Table 1. During the observation no sex related tr end with relation to diff erent food types was observed. During the micros copic observation in the lab oratory several foo d items identified from gut co ntents were recorded as. a. Fragments of aquatic plants: These types of food comprised of di fferent types of green grass, parts of seeds, leaves and root materials often found in the gut of the b oth sexes in all size groups. It ranked fifth (7%) in female and sixth (5 %) in male among foo d items during study. b. Fragment of animals: Fragments of animals were observed throughout the year during the study an d ran ked second both in female and male pra wn gut i.e. (19%) and (23%) respectively. Animal matter consisted of parts of cuticles, exoskeletons and parts of polychaetes represented by bristles, jaw, legs an d fragment of body part s, fragments of zoo planktons such as antenna, ple opods and some parts of aquatic insects their nymphs and larva. c. Algae : Fragment of green and blue green algae w ere observed throughout the study in both sexes and also in all size groups. Algae forms the third i mportant constituent of prawn food item having (19%) in female and (14%) in male. The most impo rtant genera of algae w ere Cladophora, Anabaena, Zygn ema, Volvox, Oscillataria etc . ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 788 d. Diatom: Represented mainly by Fragilaria, Pinularia, Nitzchia, and Gomphonema and r anked fourth among the food items in Macrobr achium assamense peninsular e having (8%) in male and (7 %) in female. e. Unidentified organisms: These were probably parts of the undi gested food items of both aquatic plants and animal. These were observed in the gut throughout the study p eriods and dominated in the size group of 20 -30 mm body length prawn and ranked fifth among the fo od items in male (6% ) and sixth (4%) in fe male. f. Amorphous mass: Amorph ous mass or unidentified debris comprised of fragments of decomposing organic matter which could not be accurately identified due to their tiny size an d poor physical appearance. Th is types of food items showed the dominance in gut content, i.e. male (44%) and female (41%) in Macrobrachium assamense peninsulare and was r ecorded maximum in 20- 30 mm size group in male (43.2% ) in male and (41%) in female individu al. g. Sand grains: Sand grains were found in inadequate per centage 1- 2% in the gut of both sexes throughout th e study. This item was possibly an accidental inclusion along with other food it ems, even though M. a. peninsulare is botto m feeder were mostly prefer dead and decay matter. (a) Female (b) Male Fig. 1:- Percentage of food items (%) in Macrobrachium assamense peninsular e Table 1:- Percentage co mposition of gut contents o f M. assamense peninsulare in relation to size groups ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 789 Table 2:- Monthly variation in the degree of f ullness of guts (%) in M . assamense penins ulare Discussions:- Diet of Macrobrachium assamense peninsulare is generally microscopic, containing minute animal and plant matter showing its omnivorous nature. It is evident from gut content analysis that the organic detritus (unidentified debris) appears quite regular in the die t of the Macrobrachium assamense peninsular e (Table 1) throughout the study period in different size groups although its efficacy as food source seen to be important to gr own-up prawns when it leads to benthic existence. O rganic detritus was readily consumed when the other preferred food items were less av ailable. Pringle et al. (1993) and Pringle (1996) mentioned that the de capods crustaceans are omnivores an d detritivoros. Present finding also get support from the studi es on feeding habits and food of M. acanthurus ( Albertoni et al., 2003a ), M. l amarrei ( Sharma and Subba, 2005 ), M. vollenhovenii ( Jimoh et al ., 2011 ) an d M. carcinus ( Lima et al., 2014 ) which were found to be omnivorous in nature. Quantitative estimation of gut contents of Macrobrachium assamense peninsulare is in agreement wit h the result of Raman (1967); Rao (1967); Ling (1969) and Coasta and Wanninayake (1986), who have reported that prawn are omnivores in feeding habitat. Bakhtiyar et al. (2014) also reported that the M. dayanum has been categorized as detriti-omnivo rous feeding on both animal and plant matter with detritus as dominant f ood items and similar o bservatio n was recorded in Ma crobrachium macrobrachion in Nigeria by Jos eph et al. (2013). Gut content o f Macrobrachium as samense peninsul are consisted o f fragments of a quatic plants, f ragments of aquatic animals, algae, diato ms, amorphous mass, sand grains and u nidentified organisms, whic h represented different trophic level. All types of food item were observed thr oughout the study per iod in variable proportion. In comparison to the observation of Subramanyam (1963 a) and in agreement with Gopalkrishnan (1952) , neither the size nor the sex had any r elationship with the feeding habits of th e prawn and th e similar observation was observed in our study in Rawasan prawn . Present study was also comparable with th e finding of Lee et al. (1992) analysis from gut co ntents of shrimp Lucifer faxoni . Dall (1968) had reported that the gut contents o f v arious pe naeid shrimps i.e. M. accleayi, P. plebejus, P. osculentes and P. merguiensis were mainly comprised of animal body parts (Jaw, l egs, chitin f ragments and seta) and some unidentified matters. Panikk er (1952) reported f ood of young penaeid as of tiny organism, alga and detritus. Animals are classified as herbivorous, omnivorous and carnivores on the base of food type they eat (Chopra, 1939) . Presence of fish scale by accidental entry along with detritus was observed in the gut c ontent of M. rosenbergii (R aman, 1967) and M. equidens (Murthy & Rajagopal, 1990) , whereas as n o such mate rial were reported from the g ut contents of th e Macrobrachium assamense p eninsulare during the study . Rao (1967) r eported that th e M. rosenbergii eat up their exuvia after molting and Natraj (1947) also made similar observation in P. idea. Durin g present study , we also observed M. assamense peninsulare by eating exuvia during the laborato ry inspection in aquarium and so metime in the field o bservation. Murthy an d Rajagopal (1990) recorded that in M. equidens t he most o f th e food items consist of c rustacean appendages, some parts of decay organic matter, sand a nd mud similar observations we re recorded in our study. ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 790 The per centage of empty st omach show ed an increasing trend in M . assamense peninsulare toward the end of the onset of the breeding season and it w as due to the less food intake by female prawn due t o gonadal maturation, which occupy major portion o f the cephalothorax and this is w ell compared with t he studies of M. malcolmsoni Ibrahim (1962) whe re in a reduced feeding was notice d in mature female. Acknowledgement:- The Author s’ are very much g rateful to Pr of. A. K . Dobriyal, HOD of Zoology and biotechnology, Director o f Campus Pauri, H.N.B Garhwal (A Central University), Pauri Garhwal Uttarakhand for en courageme nt and facilities provided to carry out th is w ork and also to UGC New Delhi for providing National Fellowship Fo r Higher Education to first autho r Mr. Koshal Kumar to meet the contingent expe nditure. References:- 1. Albertoni E.F., C. Pama-Silva and F.A. Esteves, (2003a ). Overlap of dietary niches and elec tivity of three shrimp species (Crustacea, Decapoda) in tr opical coastal lagoon (Rio de Janeiro, Brazil). Revista Brasileira de Zoologia , 2003a; 2091:13 5-140. 2. APHA, (2012). Standard method for the examination of water and waste water. APHA, AWWA, WPEC, 22 th edition, New York 3. Bakhtiyar, Y., R . Lakhn otra and S. L anger, (2014). Natural food and feeding habits of a locally a vailable freshwater prawn Macrobrachium dayanum (Henderson) from Jammu waters,North India. International Journal of Fisheries and Aquatic Studies ; 2(3): 33-38 4. Baskar, S. N.Narasimhan, G.Swamidass Daniel , R.Ravichelvan, M.Sukumaran, T.Anandaraj (2013). Food and Feeding Habits of Penaeusmonodon(Fabricius) from Mallipattina m Coast in ThanjavurDist, Tami l Nadu, India . International Journal o f Research in Biolo gical Sciences 3(1) : 1-4 5. Chopra, B., (1939). Some food prawns of India and their fisheries. J. Bombay N at. Hist. Soc. , 41: 221-234 . 6. Coasta, H.H. an d T .B. W anninayake, (1986). Food, feedin g a nd f ecundity of the giant fresh water prawn, Macrobrachium rosenbergi i from a natural habita t in Sri Lanka. The first Asian f isheries forum , Pp. 555-558. 7. Dall, W., (1968). Fo od and feeding of so me Australian Penaeid Shrimp. FAO F ish. Rep. 2:251-258. 8. Devi P.L., D.G. Nai r and A. Joseph, (2013). H abitat ecology and food and feeding of the herring bow crab, Veruna litterata (Fab ricius, 1798) of Coc hin backwaters Kerla India. Arthropods 2(4): 1782-188. 9. Froster, J.R.M. (1976). Studies on the development of compounded diets for prawn. Int. conference on aquaculture nutrition. Delaware sea programm e and U.S/Japan aquacultur e panel . UJNR, pp 229-248. 10. Gopalkrishnan, V., (1952). Food and fee ding habit of pe naeus indicus . J. Madras Univ ., B 22 69-75. 11. Hall, D.N.F. (1962). Observations on the taxonomy and biology of some Indo -West-Pacific Penaeidae (Crustacea, Decapoda). Fish. Publ. Colon. Off . Lo nd., (17): 229 pp. 12. Ibrahim, K.H. (1962). Obse rvations on the fishery and biology of the freshwater praw n Macrobrachium malcolmsonii of the R iver Godavari. Ind. Fish .,9(2): 433- 467 13. Jimoh, A.A, E.O. Clarke, O.O. Whenu and H.B. Adeoye, (2 011). Food an d feeding h abits of th e African ri ver prawn (Macrob rachium vollenhovenii, H erklots, 1857) in Epe Lagoon, southwest Nige ria. Int. Journal of Fisheries and Aquacultur e 2011; 3(1):10-15. 14. Joseph, I., A.E. Bass ey , E . George, U. Geo rge, (2013). FeedingHabits o f the B rackish Rive r Prawn ( M. macrobrachion, He rklots, 1857) from Great Kwa River, Obufa Esuk B each, Calabar, Cross River State, Nigeria. Journal of Natura l Sciences Research, Vol.3, No.9. 15. Khan R.A. N. A ravindan and A. Kalavat, (2 001). Distributio n of two post-larvae species of commercial prawns (Fennerope naeus indicus and Penaeus mono don) in a coastal tropical estuary. J. Aqua. Sci. 16(2): 99 – 104. 16. Lee, W., Y. Omo ri, and R. Peck, (1992). G rowth, reproduction and fee ding behavior of the pla nktonic shrimp, Lucifer faxoni Bo rraidele, off the Texas coast. Journal of Plankton Research , vo l. 14, no. 1, p. 61-69. 17. Lima, J.D.F, J.D.S. Garcia and T.C.D. S ilva, (2014). Na tural diet and feeding habits of a freshwater prawn ( Macrobrachium carcinus : Crustacea, Decapoda) in the estuary of the Amazon River. Acta Amazonica . 44(2):235-244. 18. Ling, S.W., (1969). The general biology and development of Macrobarchium rosenbergii (de man). FAO Fish reproduction 57(3) 589-60 6. 19. Marte, C. L. (19800. The food and feeding habit of Penaeus monodon Fabricius collected from Makato River, Aklan,Philippines (Dec apoda Natantia). Crustaceana , 38(3) : 225-236. ISSN: 232 0-5407 Int. J. Adv. Re s. 4(12), 7 86-7 91 791 20. Murthy, D. K. and K.V. Rajagopal, (1990). Food and feeding habits of the freshwater prawn Macrobrachium equidens (Dana). Indian J . Anim. Sci ., 60 (1): 118-122 . 21. Natraj, S. (1947). P reliminary observation o n the bionomics reproductio n and embryonic stages of Palaemon idea , Helle r. Rec Indian Mus 45 (1) pp: 89-96 22. Needham, P.R. and J.G. Needham, (1962). A guide to the study of freshwater biology 5 th (ed.) Holden Day Incorporation . 23. Panikker, N.K., (1952) . Possibilities of furthe r expansion of fish and praw n culture practices in India. Curr. Sci ., 21: 29-33. 24. Pringle C.M. (1996). 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This paper is expected to develop the data on food and feeding habits of Macrobrachium assamense peninsulare ( Figure 1) from Rawasan stream in Uttarakhand. Extensive studies on the food and feeding of Palaemonid prawn of the world have been carried out by several workers (Marte,1980;Jayachandra, and Joseph;1989, Murthy andRajagopal, 1990;Jimoh et al. 2011;Baskar et al. 2013;Lima et al. 2014; Koshal Kumar et al. 2016) . ... ... A similar pattern of variation has been noticed in the different maturity stages of Macrobrachium assamense peninsulare females after study the various models of indices related to feeding. A higher percentage of the empty stomach in females was recorded more as compared to the male individual due to the reduced feeding as a result of enlargement of the gonads before the spawning and occupies the significant portion of the cephalothorax (Koshal Kumar et al. 2016 ). GaSI of Penaeus monodon was reported by Marte, 1980 which revealed that the feeding intensity remained high during winter months and got reduced during the summer months in contradiction to the present study. ... EVALUATION OF FEEDING INDICES TO ANALYSE THE FOOD PREFERENCE AND FEEDING RATE OF MACROBRACHIUM ASSAMENSE PENINSULARE (TIWARI, 1958) FROM RAWASAN STREAM OF GARHWAL, CENTRAL HIMALAYA, INDIA Article Full-text available Dec 2020 Dr-Koshal Kumar Anitarawat Rana Chandra Bhanu Kotnala In this paper the feeding index (FI), Gastro somatic index (GaSI) and Food Prevalence Index (FPI) of freshwater prawn Macrobrachium assamense peninsulare were evaluated to assess the quantity and quality of food preferred by it in the Rawasan stream. A total of 401 prawns were collected during a span of two years from five selected sites in the Rawasan stream of Garhwal in Central Himalaya, India from August 2013 to July 2015. Collected prawns were in the size range of 20-75 mm in males and 24-65 mm in females in total length. The stomach contents of 10 collected specimens were examined monthly and observed that 35% of the stomach was full or semi-full and 15% stomach were empty. The highest Gastro somatic value was obtained 2.95±0.80 during June in the male and 3.25±0.44 during May in the female. After that, it gradually decreased in both the sexes and is repeated in cyclic patterns each year. More or less a similar trend was reported in the feeding index value during the study which indicates a significant relationship between feeding intensity and Gastro somatic index. The highest Food Prevalence Index (FPI) was seen as fragments of animal matter in both sexes, which was (88.48) in male and (58.26) in female prawns and the lowest FPI value was sand and debris (7.9 and 5.7) in male and female prawn respectively. The main food items were the fragment of plants as well as animals, diatoms, algae, and sand. Overall, the results show that this prawn is selectively abstemious. Keywords: Feeding index, Gastro somatic index, Food Prevalence Index, Freshwater prawn Studies on the Biology of Macrobrachium rosenbergii (de Man) of the Hooghly Estuary with Notes on its Fishery Article Full-text available Jan 1967 R M Rao Studies on the Biology of Macrobrachium rosenbergii (de Man) of the Hooghly Estuary with Notes on its Fishery On the record of herring bow crab Varuna litterata (Fabricius, 1798) from Cochin Backwaters, India Article Jan 2017 P. Lakshmi Devi A. Joseph Present study is on the record of occurrence of the herring bow crab, Varuna litterata from Cochin Backwaters. Study reveals that V. litterata occur in the Cochin Backwaters plentifully throughout the year and are found to be an inhabitant of mangrove regions and prefer areas with close proximity to the sea having good tidal influx. © 2017, National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved. Food and feeding habits of Penaeus indicus Article Jan 1952 V. Gopalakrishnan Observations on the fishery and biology of the giant freshwater prawn Macrobrachium rosenbergii de Man Article Jan 1967 K V Anantha Raman Observations on the fishery and biology of the freshwater prawn Macrobrachium malcolmsonii Milne Edwards in River Godvari Article Jan 1962 Standard Methods for the Examination of Water and Waste Water Article Jan 1985 M. Taras A. Greenberg R. Hoak M. Rand Standard Methods for Examination of Water and Waste Water Article Jan 1980 M.A.H. Franson Observations on the Taxonomy and Biology of Some Indo-West-Pacific Penaeidae (Crustacea, Decapoda) Article Jan 1962 D. Hall Possibilities of further expansion of fish and prawn cultural practices in India Article Jan 1952	https://www.researchgate.net/publication/312347133_QUANTITATIVE_AND_QUALITATIVE_GUT_CONTENT_ANALYSIS_OF_MACROBRACHIUM_ASSAMENSE_PENINSULARE_FROM_RAWASAN_STREAM_OF_GARHWAL_HIMALAYA_UTTARAKHAND_INDIA
Remote Sensing \| Free Full-Text \| Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation The camera characterization procedure has been recognized as a convenient methodology to correct color recordings in cultural heritage documentation and preservation tasks. Instead of using a whole color checker as a training sample set, in this paper, we introduce a novel framework named the Patch Adaptive Selection with K-Means (P-ASK) to extract a subset of dominant colors from a digital image and automatically identify their corresponding chips in the color chart used as characterizing colorimetric reference. We tested the methodology on a set of rock art painting images captured with a number of digital cameras. The characterization approach based on the P-ASK framework allows the reduction of the training sample size and a better color adjustment to the chromatic range of the input scene. In addition, the computing time required for model training is less than in the regular approach with all color chips, and obtained average color differences & Delta; E a b * lower than two CIELAB units. Furthermore, the graphic and numeric results obtained for the characterized images are encouraging and confirms that the P-ASK framework based on the K-means algorithm is suitable for automatic patch selection for camera characterization purposes. Line Spacing: Column Width: Background: Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation by Adolfo Molada-Tebar 1,,†,‡ , Ángel Marqués-Mateu 1,†,‡ , José Luis Lerma 1,†,‡ and Stephen Westland 2,†,‡ 1 Department of Cartographic Engineering, Geodesy, and Photogrammetry, Universitat Politècnica de València, 46022 Valencia, Spain 2 Colour Research Group, School of Design, University of Leeds, Leeds LS2 9JT, UK Author to whom correspondence should be addressed. † Current address: Camino de Vera s/n, Edificio 7i, 46022 Valencia, Spain. ‡ These authors contributed equally to this work. Remote Sens. 2020 , 12 (3), 520; https://doi.org/10.3390/rs12030520 Received: 17 January 2020 / Revised: 27 January 2020 / Accepted: 4 February 2020 / Published: 5 February 2020 (This article belongs to the Special Issue Remote Sensing, Metric Survey and Spatial Information Technologies for Heritage Management ) Abstract The camera characterization procedure has been recognized as a convenient methodology to correct color recordings in cultural heritage documentation and preservation tasks. Instead of using a whole color checker as a training sample set, in this paper, we introduce a novel framework named the Patch Adaptive Selection with K-Means (P-ASK) to extract a subset of dominant colors from a digital image and automatically identify their corresponding chips in the color chart used as characterizing colorimetric reference. We tested the methodology on a set of rock art painting images captured with a number of digital cameras. The characterization approach based on the P-ASK framework allows the reduction of the training sample size and a better color adjustment to the chromatic range of the input scene. In addition, the computing time required for model training is less than in the regular approach with all color chips, and obtained average color differences Δ E a b * lower than two CIELAB units. Furthermore, the graphic and numeric results obtained for the characterized images are encouraging and confirms that the P-ASK framework based on the K-means algorithm is suitable for automatic patch selection for camera characterization purposes. Keywords: archaeology ; clustering ; colorimetry ; data mining ; machine learning ; rock art documentation 1. Introduction The risk factors that endanger the integrity of cultural heritage have risen at alarming rate during the last decades. In addition to the natural erosion or weather events, other effects caused by climate change or some human acts of vandalism or terrorism must be considered [ 1 , 2 ]. Especially vulnerable are those heritage items located in open-air emplacements such as archaeological rock art painting caves. Thus, it is not surprising that the scientific community, particularly the heads of cultural heritage conservation, and society in general, are very sensitive about the potential threats that endanger our historical places. Exhaustive and precise documentation is vital in order to adopt urgent and concrete actions to preserve our heritage objects, especially those in danger. To address this priority task, traditional methodologies should be supported by new state-of-the-art techniques. In this respect, the integration of geomatics techniques such as photogrammetry, remote sensing or 3D modelling are indispensable at present, and has been a considerable step forward for new cultural heritage applications [ 3 ]. Previous examples of such applications include 3D photogrammetric and laser scanning documentation [ 4 , 5 , 6 ]; automatic change detection techniques [ 2 ]; monitoring for deformation control [ 7 ]; microgravimetric surveying technique [ 8 ] or imaging analysis [ 9 , 10 ] or enhancement methods [ 11 , 12 ] to name just a few. The use of novel technologies allows users to solve aspects such as the graphic and accurate metric archaeological documentation, usually in a fast and effective way. However, there are still issues to be addressed, such as the precise specification of color which is an essential attribute in cultural heritage documentation. An accurate measurement of color allows the current state description of historical objects, and gives trustworthy information about its degradation or aging over time [ 13 ]. Precise color recording is a priority, yet not trivial, task in heritage documentation [ 14 , 15 ]. Traditional methodologies for color description in archaeology are mostly based on visual observations which are strictly subjective. In order to obtain precise color data it is necessary to rely on non-subjective and rigorous methodologies based on colorimetric criteria. Although current cultural documentation tasks increasingly rely on digital images techniques, it is well known that the color signal recorded by the camera sensor (generally as RGB format) are not colorimetrically sound [ 16 ]. The digital values acquired by the sensor are device dependent; that is, different camera sensors will record different responses even for the same scene under identical lighting conditions. Moreover, the RGB data do not directly correspond with the independent, physically based tristimulus values based on the Commission Internationale de l’Éclairage (CIE) standard colorimetric observer, nor do they satisfy the Luther–Yves condition [ 17 ]. Therefore, a rigorous processing framework is still necessary in order to collect color data using a digital device in a meaningful way. A common and acceptable approach is by means of digital camera characterization [ 18 , 19 , 20 ]. The fundamental goal of the characterization procedure is to determine the mathematical relationship between the input device-dependent digital camera responses and the output device-independent tristimulus values of an input image scene. Thus, as a result of camera characterization, the tristimulus values for the full scene can be predicted on a pixel-by-pixel basis from the original digital image values captured [ 21 ]. Some factors to consider during the characterization procedure are: the camera built-in sensor, camera parameters during the photographic acquisition phase, size and color properties of training data [ 22 , 23 ], and the regression model used [ 24 ]. The sensor has an effect not only in terms of noise but in the characterized output images [ 25 ]. Thus, the interpolation method selected must be properly adapted to the data offering a suitable graphic solution. The number of color patches measured and their distribution also plays a decisive role [ 26 ]. The manufactured color charts are designed to cover the maximum range of imaged colors on photographic or industrial applications. However, in most applications it is not required to use the entire set of color patches as a training set since redundant data could be entered into the regression model. In addition, color charts are not developed specifically for archaeological purposes, where only a few colors are present on natural scenes. Thus, an adequate selection of training samples for the regression model is crucial for quality estimations [ 22 , 27 , 28 ]. Our previous studies showed that accurate results can be achieved using a reduced number of properly selected samples [ 24 ]. Accordingly and following the lines established in our previous investigations, in this paper we propose a novel framework for an optimal color sample selection of training data. The algorithm is based on a K-means clustering technique. We call this procedure Patch Adaptive Selection with K-means or P-ASK. The use of the P-ASK framework allows us to extract the dominant colors from a digital image and to identify their corresponding chips in the color chart used as a colorimetric reference. The aim of the P-ASK dominant color patch selection is to carry out the characterization with this color sample set, instead of using the common approach based on the whole color chart. In the P-ASK approach, it is possible to reduce the number of training samples without the loss of colorimetric quality, using the most representative samples related within the chromatic range of the input scene. However, the main contribution of this paper is not only the framework for the K-means methodology but the practical assessment of the results, specifically for recording and documenting properly the color of the rock art scenes. Several methodologies can be found in the literature about optimal color sample selection, such as those based on metric formulas [ 27 ], adaptive training set [ 26 , 29 ], or minimizing the root-mean-square (RMS) errors [ 30 ]. OlejniK-Krugly and Korytkowski developed a procedure based on ICC profiles using a standard ColorChecker and a set of custom color targets by means of direct measurements on the artwork [ 31 ]. Clustering analysis is widely applied for the extraction of representative samples [ 28 , 29 , 32 , 33 ]. Eckhard et al. proposed a clustering-based novel adaptive global training set selection and perform the comparison between different well established global methods for colorimetric quality assessment of printed inks [ 28 ]. Particularly, Xu et al. applied a K-means clustering algorithm in multispectral images to extract a set of training samples directly from the art painting for spectral image reconstruction applications [ 33 ]. These studies showed that better results were achieved using optimal color samples. We tested the P-ASK framework proposed in this paper on a set of rock art scenes captured with different digital cameras. The characterization approach with dominant colors offered numerous advantages as reported in the following sections. The rest of the paper is structured as follows. In Section 2 , we describe in detail the theoretical basis and the equipment required to perform the P-ASK framework proposed. Section 3 contains the results obtained from the processing of laboratory images and rock art scenes. Finally, in Section 4 and Section 5 we present the discussion and the main conclusions of this research. 2. Materials and Methods 2.1. Study Area: La Cova dels Cavalls Rock Art Shelter Cova dels Cavalls (“The cave of horses”) is one of the most singular rock art archaeological sites of the Mediterranean Basin on the Iberian Peninsula. It is part of the Valltorta complex, located in Tírig, Castellón, in eastern Spain ( Figure 1 ). This cave is one of the set of rock art sites inscribed in the UNESCO World Heritage list since 1998 [ 34 ]. Figure 1. Location map of Cova dels Cavalls rock art Site (Source: Institut Cartogràfic Valencià [ 35 ]). 2.2. Data Set Acquisition The image set contains the so-called main hunting scene, which shows a group of hunters shooting arrows with their bows to a herd of deer ( Figure 2 ). It is undoubtedly a vivid and spectacular human-centered scene, filled with movement, where the use of reddish pigment dominates [ 36 ]. Figure 2. Rock art images: ( a ) Fujifilm IS PRO ( b ) Sigma SD15 ( c ) Nikon D40. Three different Single Lens Reflex (SLR) cameras were used in this study for image acquisition: a Fujifilm IS PRO with a Super CCD sensor (23 × 15.5 mm); a Sigma SD15 with a CMOS Foveon®X3 sensor (20.7 × 13.8 mm); and a Nikon D40, with a CCD sensor (23.7 × 15.6 mm). All those cameras allow taking pictures in a RAW format, which gives better results than processed images after characterization [ 20 , 21 ]. In fact, the use of RAW images allows us to disable the activation of the set of processing operations applied to the original images, such as white balancing, demosaicing or gamma correction, which implies the modification of the original data acquired by the sensor [ 20 , 37 ]. A main difference across cameras is their built-in image sensors. While the Super CCD sensor (Fujifilm IS PRO) and CCD sensor (Nikon D40) are monochrome with color filter array (CFA) and Bayern pattern for color interpolation, the Foveon®X3 sensor (Sigma SD15) is trichromatic, that is, it registers the color values for each channel separately without interpolation [ 38 ]. The scenes in the digital images should contain a chart as colorimetric reference. In our study we chose the X-rite ColorChecker®Digital SD chart, which is widely used in digital image processing pipelines. Although this color chart has 140 patches, we used only the 96 chromatic central chips (and 15 grey scale central ones) without considering the external patches on the edges. The RAW RGB data for these patches were extracted from the images using our own software named pyColourimetry [ 39 ]. The spectral reflectance data of the color patches from the ColorChecker were measured directly using the spectrophotometer Konica Minolta CM-600d. A total of 96 patches were measured under the CIE recommendations: 2° standard observer, and D65 illuminant. The spectral data collected can be easily transformed into CIE XYZ tristimulus values using well-known CIE formulae [ 40 ]. 2.3. Colorimetric Camera Characterization Procedure Image-based camera characterization is a well-known procedure described in numerous references [ 18 , 19 , 20 , 21 , 41 ]. The main stages of the imaged-based camera characterization procedure are (1) Training: sets the training data for model learning, (2) Regression model fitting and checking, and (3) Image characterization: creates the output sRGB image ( Figure 3 ). In the following subsections, we provide detailed descriptions of each step. Figure 3. Colorimetric camera characterization framework. 2.3.1. Training Data Image-based characterization procedures rely on the training data set that typically permits model learning via the computation of the parameters of the selected mathematical function. The regression model is developed on the basis of the original data available: the camera responses (RAW RGB) and their colorimetric coordinates (CIE XYZ), both from the reference color chart. As for the comparative analysis, we will separate two subsets of training data from each original dataset: the whole color patches (Full ColorChecker or FCC data set), and the selected dominant patches from the P-ASK framework (K-means dominant patches or KDP data set). Therefore, we will get two independent outcomes as outlined in Figure 3 ). 2.3.2. Regression Model and Accuracy Assessment The image-based characterization procedure used in this study is based on an empirical regression model which defines the relationship between the input and output spaces. Different regression models have been used for camera characterization purposes, including polynomial models [ 41 ], artificial neural networks [ 42 ], polyharmonic splines [ 26 ] or Gaussian processes [ 25 ] to name a few. However, in recent years, research has focused on spectral recovery techniques [ 43 ]. In this paper, we used a classical second-order polynomial regression model, since it is computationally simple and gives valid results for archaeological applications [ 21 , 25 ]. In terms of good practice, the training data used for learning usually should not be used in model assessment since it may result in an overly optimistic prediction model error estimation. Instead, it is preferred to set aside an independent test data set used exclusively for model evaluation. Alternatively, a cross-validation framework may also be used, where the training data is repeatedly separated into training and testing sub data sets. In this study, a leave-one-out cross validation (LOOCV) procedure was performed for model accuracy assessment [ 44 , 45 ]. Two different metrics were used for the quality evaluation: the CIE XYZ residuals for model checking, and the color difference values in the colorimetric assessment of the characterized images. Regarding the color difference computation we used the Δ E a b * formula [ 40 ] which performs well in rock art measurement conditions [ 25 ]. In colorimetric accuracy studies, it is usual to establish a threshold value known as “Just Noticeable Difference” (JND) or tolerance for Δ E a b * . In this study, we set a JND value of four CIELAB units which ensures imperceptible variations to human vision [ 46 ]. 2.3.3. Output sRGB Characterized Image An additional transformation is required in order to reproduce the output CIE XYZ values on display or printing devices. We followed the International Electrotechnical Commission (IEC) specifications to transform the CIE XYZ tristimulus values into the sRGB color space, which is compatible with most digital devices [ 47 ]. 2.4. K-means Clustering Technique K-means is a center-based clustering algorithm widely used in different applications of data mining, machine learning and pattern recognition [ 48 ]. K-means aims to minimize the mean squared distance from each N-data point to its K-nearest cluster center, following four basic steps to: (1) set initial K cluster centers, (2) group samples in the K clusters, (3) recompute the new K cluster centers, (4) iterate the process until the algorithm converges [ 49 , 50 , 51 ]. Since the introduction of the K-means algorithm in its current form [ 48 ], different alternatives and improvements to achieve better performance have been published, including an algorithm less sensitive to initialization settings [ 50 ] or filtering algorithms [ 52 ]. In this study, we developed a Python script to apply the K-means algorithm to our specific problem, which is to select dominant colors in camera characterization. The Python script leverages the Scikit-learn package (v0.21.3) [ 53 ] which includes an optimized implementation of the K-means algorithm together with others machine learning algorithms for supervised and unsupervised classification applications [ 53 , 54 ]. Particularly, we used the KMeans function (see [ 55 ] for more details). However, before applying the K-means clustering algorithm, it is required to take into account some quirks associated with this technique. K-means yields accurate results when the K number of clusters and its initial centroid points are properly defined. Moreover, as an iterative technique, the K-means algorithm is potentially sensitive to initial starting conditions [ 56 ]. Thus, an inappropriate determination of initial parameters could highly decrease the accuracy of the results [ 57 ]. In our study, the inappropriate selection of clusters means that the results will not match with the real dominant colors present in the image. Therefore, it is necessary to overcome the negative effects derived from K-means initial approximations by properly setting the K number of clusters for optimal implementation of the algorithm and proper determination of the initial values. 2.4.1. Optimal K Number of Clusters The first issue to address is how to set the optimal K number of clusters and their corresponding initial centroids, since the K clusters should be known beforehand and specified by the user. Several factors could affect the selection of K such as the level of detail, the internal distribution of the samples values or its interdependence with other objects [ 49 , 58 ]. Different methods can be applied for selecting the number of clusters, such as those based on automatic process [ 57 ], probabilistic clustering approaches or visual verification [ 58 ]. To determine an appropriate number of clusters for K-means clustering, a trial-and-error process experiment was conducted. We used the rock art scene images as input data to evaluate the processing time for different values of K. In general, K-means clustering is used not only to determine the natural groups in the data, but also to identify irregularities in their distribution. However, in rock art scenes the data tends to be uniform, and we expect a set of dominant colors made of a reduced number of instances, which in turn will be highly correlated. In order to determine a proper value of K, we assessed the processing time for values of K in the range 10–100. 2.4.2. Clustering Algorithm The basic K-means algorithm [ 48 ] has been updated to improve its effectiveness, robustness and convergence speed [ 50 ]. Some of the most widely known implementations are: Forgi [ 59 ], expectation-maximization or EM-Style algorithm [ 60 , 61 ], Standard algorithm (LLoyd’s algorithm or Voroni iteration) [ 62 ], and Elkan [ 63 ]. Two of those algorithms are implemented in the Scikit-learn KMeans function: EM-style for sparse data and Elkan for dense data. Users can specify their choice via the “algorithm” parameter, using “full” or “elkan” for the EM-style or Elkan option respectively (see an example of code in Section 2.5.1 for syntax details). Given that our study is based on moderately large data sets from the RGB triplets of the digital image, the optimal algorithm to compute K-means is Elkan. The Elkan algorithm is an optimized version of the standard K-means clustering method based on the triangle inequality. The aim is to reduce its processing time by avoiding unnecessary distance calculations [ 63 ]. 2.4.3. Initial Starting Conditions The K-means algorithm searches for the optimal data partition into the K clusters established by the user. Thus, K-means provides a set of K point clusters defined by their centroids, and assigns every data point into its nearest cluster centroid. However, the final group partition is highly dependent on the initial cluster centers. Since the centroids of the clusters are usually unknown in advance, the K-means algorithm relies on a random process to set the starting cluster centroids, which are frequently chosen uniformly on the range of the data [ 56 ]. The random initialization method takes a set of K arbitrary centers uniformly selected at random as starting points. The drawback of pure random selection is the high number of iterations required to converge, and furthermore different runs with the same data do not give exactly the same results. In order to overcome this limitation, the K-means++ initial method was developed to improve algorithm speed and give more consistent results despite being a random method [ 64 ]. In fact, this is the default option for the KMeans function. The lack of consistency of random methods has been the subject of previous research. However, initialization methods based on randomness are considered as a proper selection procedure that gives satisfactory results [ 49 ]. In this study we run a simple experiment to find out the optimal initial values based on random algorithms. We just run the algorithm several times which has been reported as a suitable method to account for randomness issues [ 56 ]. We used the Scikit-learn K-means++ initial method, since it is a optimized and fast random process [ 64 ]. This analysis will allow us to determine whether or not the algorithm consistently provides the same dominant color patches although being based on a random process. 2.5. Color Patch Adaptive Selection with K-Means (P-ASK) The aim of the P-ASK procedure is to establish a methodology for the automatic selection of color training samples for camera characterization, and keep the process as simple as possible. The P-ASK framework can be separated into two main stages: the application of the K-means algorithm for the dominant color extraction of the input image, and the identification of dominant color patches as training data for the camera characterization. The first stage of this procedure can be considered as an specific image segmentation application, since it is intended to group the pixels of an image according to their color similarity. The main steps in P-ASK framework ( Figure 4 ) are: (1) create a white balanced image (D65 illuminant), (2) select support and pigment samples, (3) extract K-means dominant colors, and (4) identify dominant color patches. Further detailed explanations about those steps are given in the subsections below. Figure 4. P-ASK framework. 2.5.1. K-means Dominant Color Extraction The first aim of P-ASK is the dominant color extraction from a digital image. Previously to applying the K-means clustering algorithm two basic operations are required: white balance and sample selection. The CIE XYZ data were acquired under the D65 illuminant specification. Thus, in order to find the patches corresponding to the dominant colors, the RGB values should be strictly registered under the D65 illuminant. Since usually it is not possible to work under controlled illuminant conditions in open-air cultural heritage sites such as rock art archaeological shelters, we used a white balanced image under the hypothesis of D65 illuminant as an approximation to natural daylight. From an original RAW image, it is possible to obtain a white balanced image using the rawpy Python package [ 65 ]. One problem associated with the application of the K-means clustering algorithm to full images is the significant increase of the convergence time, given the large size of the data set. A simple and suitable option for our framework is to reduce the image size to compute only a selected image samples instead of full images. For rock art applications, the main interest is focused on defining the representative colors, especially of the pigments an the main support colors. Thus, we selected a set of samples from significant support and pigment areas for the K-means computation ( Figure 5 ). Figure 5. Support and pigment samples: ( a ) Nikon D40 image ( b ) Sigma SD15 image ( c ) Fujifilm IS PRO image. Finally, the digital data are passed into the algorithm as RGB triplets (nRGBdarray) for subsequent computing. The Python code line which runs the K-means algorithm is as follows: clt = KMeans(n_clusters = K, init = " K-means++ " , n_init = 10, max_iter = 300, tol = 0.0001, precompute_distances= " auto " , verbose=0, random_state = None, copy_x = True, n_jobs = None, algorithm = " elkan " ).fit(nRGBdarray)} where the main parameters introduced to the algorithm are: the K number of clusters; Elkan as the algorithm since it works well for dense data; and the K-means++ initialization method. The remaining parameters have been assigned to the default values of the Scikit-learn KMeans function [ 55 ]. 2.5.2. Identification of Dominant Color Patches The outcome of the K-means algorithm consists of a set of centroids, one for each K cluster. Those coordinates are a list of K RGB triplets of dominant colors, which represent the main colors found in the input digital image. The second stage seeks the correct identification of the color chart patches from the dominant colors from the K-means algorithm. The dominant colors extracted in the RGB color space can be easily transformed to CIE XYZ tristimulus values [ 40 ] and CIELAB coordinates [ 66 ] as required for subsequent processing. With the CIELAB coordinates the color differences are computed between each dominant color and the whole set of color chips. It is then possible to identify the nearest patches to each dominant color found in the image by looking for the lowest Δ E a b * . For the identification of color patches, four different approaches are tested after varying the input data and colorimetric criteria: (1) using the n nearest patches found in the whole image (NN), (2) removing the color chart from the scene and identifying the n nearest patches (RC), (3) using a set of samples corresponding to pigment and support classes and creating a new, joined synthetic image (JS), and (4) using the same sample set used in (3), but computing each class separately (SS). Note that while in the first method we do not specify any colorimetric criteria, in the remaining cases we search for the nearest patches to each dominant color with Δ E a b * color differences less than 10 CIELAB units (maximum value allowed in cultural heritage guidelines [ 46 ]). Although the use of the nearest patches extracted from dominant colors using K-means offer accurate colorimetric results, it does not always imply a proper output characterized image, that is, an image with true colorimetric sense. Our experience shows that a set of minimum fixed patches should be included in order to obtain accurate and colorimetrically sound characterized images [ 24 ], including the primary additive colors [E4, F4, G4] and the basic grayscale chips [E5, F5, G5, H5, I5, J5] (highlighted in Figure 4 , step number 4). 3. Results In this Section we describe all processing tasks, together with their output results, that support some practical considerations to define the P-ASK. Specific tests were run to define the optimal parameters of the algorithm and to assess its performance in controlled laboratory conditions as well as in rock art sites with uncontrolled light conditions. 3.1. Setting the Optimal Parameters for the K-Means Clustering Algorithm As reported in Section 2.4 , several factors could affect the results of the K-means clustering method, namely the K number of clusters and centroids to generate, the input size image, the initialization method and the algorithm applied. 3.1.1. K Number of Clusters The first issue to consider is the proper determination of the K number of clusters. That number can be determined by simple heuristics, for instance by testing several values of K and taking some convenient value, or with specific data mining tools such as the Elbow [ 67 , 68 ] or the Silhouette graphic methods [ 69 ]. A preliminary experiment was conducted under the worst-case scenario; that is, using the whole image as input data which gives some sort of upper bound in terms of computation time. The results about the running time required for computing K-means clustering are displayed in Table 1 . According to those results, K = 24 seems to be an acceptable number of clusters. The choice of this value, however, is based on the running time experiment only, and thereby it is somewhat arbitrary. It is timely, then, to confirm our choice by other means, with a consensus across numerical results, graphical tools and colorimetric criteria. Table 1. Evaluation of K-means running time with different values of K. From a color viewpoint, our study aims at characterizing digital cameras properly with a reduced number of color chips whether it is possible, which is in many ways is rather the same as performing color quantization of the image. In addition, our application field, the recording of Levantine rock art archaeological shelters requires a very specific color domain, mostly in the chromatic range of red, brown, and yellow colors which in turn are little saturated (check dominant colors extracted in rock art images in Section 3.3 ). Thus, a number of 24 seems a reasonable choice, provided that output images are rigorous duplicates of the real specimens ( Section 3.4 and Section 3.5 report on this point). In addition to colorimetric criteria, we tested the Elbow and Silhouette methods which provide graphic aid to determine the right K number in clustering problems. The Elbow method [ 67 , 68 ] consists of running the K-means algorithm on the dataset for a range of values of K, and calculate the distortion and inertia values for each K. The distortion value is the average of the Euclidean distance between all the cluster centroids for each K; the inertia is calculated as the sum of the Euclidean distances to their nearest cluster centroids. Those parameters are useful to describe how close the different K clusters are to each other. Clusters that are too close may be an indication of incorrect data partitioning, so that they could be integrated into the same cluster. Figure 6 shows the plot of the Elbow analysis using the inertia criterion, which exhibits a sharp change in slope around K = 24 (cf. vertical red line on Figure 6 ) and linear behavior beyond this K value. This result is compatible with the previous running time test ( Table 1 ). Figure 6. Elbow method using inertia. The silhouette analysis is another useful tool to analyze the separation distance among cluster centroids, which ultimately reports on the quality of the data partition. In this method, each K cluster is represented by its so-called silhouette, which is calculated as a result of the comparison between its tightness and separation [ 69 ]. The mean silhouette width, also known as silhouette coefficient or silhouette score, provides an evaluation of clustering validity, with a value within the range [−1,1]. Negative values indicate wrong cluster assignment, whereas positive values (the closer to 1, the better) indicate well-separated neighboring clusters. We run the Silhouette analysis using the Scikit-learn module which implements the method in two functions called metrics.silhouette_score and metrics.silhouette_samples. Both functions require the output of the KMeans function as input parameter [ 70 ]. Figure 7 displays the graphic results given by the Silhouette method for the Fujifim IS PRO image with K = 24. All silhouette coefficients are similar (around 0.6) with narrow fluctuations, which is an indicator of good clustering for the chosen number of clusters K [ 71 ]. Furthermore, all coefficients are above the average silhouette score (vertical red dashed line in Figure 7 ) which confirms the value 24 as a proper choice for clustering. The silhouette method also provides a graph for visualizing the clustered data ( Figure 7 b), which shows a very dense, but well separated, data set as expected in our case study. Figure 7. Silhouette analysis for the Fujifilm IS PRO. 3.1.2. K-means Initialization Once the K number of clusters is determined, the next step is the proper selection of the algorithm and the initialization method to run K-means. The KMeans function in the Scikit-learn Python package provides two algorithms: EM-style and Elkan. Since we usually work with large, dense data sets collected from digital images, we chose the Elkan algorithm which is known to perform well in such circumstances [ 55 , 63 ]. The second parameter to be determined is the initialization method of the algorithm. As usual in all implementations, the KMeans function relies on a pseudorandom parameter to start the process. Theoretically, the best option in the KMeans function is the K-means++ method, which is an optimized and fast, but still random approach [ 55 ]. A random initialization is a convenient approach to create self-starting methods and reduce user intervention. However, it has the potential drawback of returning different or inconsistent results in different runs [ 57 , 58 ]. This characteristic is clearly against common scientific and engineering conventions such as repeatability or reproducibility, and its effects on the results must be verified. In our study, the K-means algorithm was run multiple times (100 realizations) to find out whether or not the K-means reported the same set of dominant colors in a consistent manner. We then grouped the results by dominant color and created a list of RGB triplets for each dominant color found. Every list was processed to compute basic statistics: mean, minimum, maximum, standard deviation, coefficient of variation, and so on. Those results confirmed that the K-means algorithm reports dominant colors very consistently. The RGB differences within every dominant color were all negligible which also applies when we computed the Δ E a b * differences of the data ( Figure 8 ). All color difference values were less than 1 CIELAB unit ( Δ E a b * < 1, red line on Figure 8 a), clearly under the JND threshold value. The standard deviation error was less than three digital level units ( Figure 8 b). Furthermore, and most importantly, the set of colors is consistently in the same range, which confirms the suitability of the K-means algorithm to determine dominant color in rock art scenes for camera characterization purposes. Figure 8. Randomness assessment: ( a ) Δ E a b * color differences ( b ) Standard deviation error. 3.1.3. Identification of Dominant Color Patches The input image size, that is the data set size, has a direct effect on the processing time required for the K-means algorithm ( Section 3.1.1 ). A simple solution to reduce the processing time is to use small sample images instead of the complete picture. However, our study seeks an efficient methodology not only in terms of computation speed, but mostly regarding its performance in identifying color patches. In this line, four different methods were tested prior to setting the optimal path for identifying color patches. Table 2 shows the results of each method. Those four methods are briefly described as follows: Table 2. Comparison of four methods to identify dominant color patches. Nearest n-patches (NN). The complete image is processed. Remove ColorChart (RC). The color chart is removed from the original image. Joined samples (JS). Pigment and support samples are merged in a new synthetic data set and processed together. Split samples (SS). Pigment and support samples are processed using two different data sets. The NN method detected the highest number of patches using the four nearest patches (n = 4). It should be noted, though, that there are no colorimetric restrictions in this approach, which means that some patches can be out of the range of dominant colors extracted from the images or, in other words, some patches may not fit the real rock art scene. This point came out when observing some visual inconsistencies between the patches detected by the algorithm and the scenes under study. The other methods detected less patches but returned better colorimetric results. This was the expected result since colorimetric constraints somehow guarantee better fit of the selected patches to the scene. The RC method systematically gave the smallest number of patches and should be rejected since the other three methods provide similar results regardless of the camera sensor in use. The method that offered the best results was the SS, therefore, splitting samples for the pigment and the support areas seems to be the most reasonable choice. We also conducted a detailed analysis of the SS method with focus on performance as a function of the number of clusters K ( Table 3 and Figure 9 ). According to these results, the number of color patches detected stabilizes, whereas the processing time grows linearly without improving the collection of color patches. Therefore, based on these results, the best option seems to be the SS method with K = 24. Figure 9. Graphic results for the SS method: ( a ) Patches identified; ( b ) Processing time. Table 3. Assessment of the SS method for the identification of dominant patches. 3.2. Test on the DigiEye Imaging System Before applying the P-ASK framework on rock art images, several tests were run in a laboratory environment under highly controlled light conditions. We used the DigiEye imaging system [ 72 ] which allows taking pictures in an optimal environment to conduct colorimetry experiments. The images were captured with the SLR Nikon D40 camera and the color reference was the X-rite Passport color chart under the D65 illuminant ( Figure 10 ). Figure 10. DigiEye imaging system: ( a ) DigiEye system (without camera integrated) ( b ) Nikon D40 test image (D65 illuminant). The test presented in this Section is a reduced version of the whole P-ASK method. In the test we obtained images of the color checker, without rock art specimens, which provides some sort of “benchmark environment” to assess the quality of P-ASK ( Figure 11 ). Figure 11. P-ASK framework adapted to the DigiEye D65 image acquired with the Nikon D40. The input image was a clipped picture of the X-rite color checker and the working parameters were: K = 25 (24 colored patches and one color for the background), Elkan algorithm, K-means++ initialization, and the SS method for the identification of the dominant patches. Figure 11 shows the performance in the identification of colors on the chart. Our method can perfectly identify all colors present in the chart without any missing patches. This result is certainly compelling and shows the potential of P-ASK to adapt to the whole chromatic domain of the scene, which makes the method a suitable choice to be applied in documenting rock art images. 3.3. Assessment of the P-ASK Framework on Rock Art Images Since the P-ASK framework provides good results in laboratory conditions, the next step is the assessment using images collected in rock art shelters. In order to simplify this paper, we present the results for the Fujifilm IS PRO image only. We should note that all steps described in Figure 4 are carefully computed, using the parameters established in previous sections: K = 24, Elkan algorithm, K-means++ initialization, and SS method. We emphasize once again that the aim of the P-ASK framework is to (a) extract the dominant colors present on the rock art scene, (b) identify its corresponding patches in the color chart, and (c) carry out the characterization using those patches as training data. From the Fujifilm IS PRO white-balanced image, a set of twelve samples were collected for the extraction of dominant colors, five samples for the support (S1, S2, S3, S4 and S5), and seven for the pigment (P1, P2, P3, P4, P5, P6 and P7). Figure 5 c shows the sampling areas that were clipped to get the samples for subsequent processing. Figure 12 contains a detailed description of pigment sample P1 consisting of the detail clipped image, the color palette of the dominant colors, and its frequency histogram. P-ASK provides this information for each sample separately. Figure 12. Graphic description of the pigment sample (P1): ( a ) clipped detail image, ( b ) K-means centroids, ( c ) Dominant color palette, ( d ) Dominant color frequency histogram. The last step prior to camera characterization is to select a list of patches from the color chart that match with the dominant colors obtained from the samples. The results provided by P-ASK consist of 9 patches for the support and 14 for the pigment (see a summary in Table 4 and Figure 13 ). This is the expected result considering that the support rock material is highly uniform and requires less colors to be characterized. Note also that duplicates appear across samples of the same class (either pigment or support) as well as across samples of different classes. This characteristic is also expected since the sampling areas for pigment characterization also contain some amount of support. Figure 13. Color patches detected: ( a ) Support samples; ( b ) Pigment samples; ( c ) Support-pigment; ( d ) KDP training samples. Table 4. Color Patches detected from the Fujifilm IS PRO samples. A total of 15 color patches are identified from the chromatic image information with P-ASK ( Table 4 and Figure 13 c). We have to note that none of the previous methods integrate “color insight” in their rationales. It is the user’s responsibility to append such insight, as we did when extending the set of color chips with three primaries and six grays. Thus, the KDP training dataset for the Fujifilm IS PRO camera consists of a total of 24 color patches (cf. Figure 13 d). 3.4. Model Accuracy Assessment of the Image-based Characterization Two different sets of training data are used to build the regression model used in the camera characterization procedure: the FCC set (96 samples) and the KDP (24 selected reported by P-ASK). We conducted a comparison between the results achieved for both training data sets. Table 5 shows the basic statistics of the LOOCV residuals of the CIE XYZ predicted values after model adjustment. For the sake of completeness, we also include the residuals of the FCC model applied to the dominant patches in the KDP model, which helps in comparing all data sets. The histograms of the LOOCV CIE XYZ residuals are also displayed in Figure 14 . Figure 14. LOOCV CIE XYZ Residuals histogram: ( a ) FCC (24 patches) ( b ) KDP (24 patches). Table 5. CIE XYZ Residuals of the FCC and KDP models. By comparing the residuals of the FCC (with 24 patches) and the KDP model, we find slightly better results in the KDP model, especially in the minimum and the standard deviation statistics ( Table 5 , Figure 14 a,b). Although the assessment of residuals is very useful to evaluate the predictive capability of the regression model, the computation of the Δ E a b * color differences is required in colorimetry experiments to rigorously analyze the accuracy achieved after camera characterization. Table 6 shows basic color difference statistics computed for the FCC and KDP models. Once again, the Δ E a b * values obtained by the KDP model show an improvement in terms of colorimetric accuracy when compared with the FCC model. The better results given by the KDP model can be seen graphically in the one-to-one color patches plot displayed in Figure 15 . All Δ E a b * in the KDP samples were lower than in the FCC samples, and always under the JND threshold value fixed in 4 CIELAB units (cf. Figure 15 a,b). Figure 15. Δ E a b * color differences: ( a ) FCC (24 patches); ( b ) KDP (24 patches). Table 6. Δ E a b * color differences in the FCC and KDP characterization experiments. 3.5. Output sRGB Characterized Images Ideally, the final outcome obtained through the characterization process is an image with “colorimetric characteristics.” A common approach to preserve such characteristics in digital pictures is the use of the sRGB space, where the RGB data are defined in a physically based, device-independent color space via transformation functions of CIE XYZ tristimulus values [ 47 ]. The output sRGB characterized images are shown in Figure 16 . By observing those sRGB images, it is evident that the results are very satisfying in visual terms, regardless of the training data used for the regression model. Figure 16. Output sRGB characterized images: ( a ) FCC training data (96 patches) ( b ) KDP training data (24 patches). Although the comparison between the two models confirms that the KDP model performs better in numerical terms, the images provided by the two different training datasets show that both characterization approaches transform successfully RGB data to the CIE XYZ independent color space. It is challenging, even for experienced observers, to perceive any visual differences between the two output images (cf. Figure 16 a,b). In addition to the numerical computations, we created a Δ E a b * mapping image with the color differences between the characterized images obtained with the FCC and the KDP training data sets ( Figure 17 ). The predominant green color present on the difference mapping image indicates that the values of color difference values are less than 2 CIELAB units (see map key in Figure 17 ), which is a very good result in colorimetric terms. Actually, we found higher differences in two cases corresponding to (a) cracks in the rock, where hard shadows are produced (yellow pixels with differences between 2 and 3 units), and (b) some patches of the color chart out of the color domain of the scene (differences greater than 2 units coded with yellow, orange and red pixels). Since the focus of the study is on accurate colorimetric analysis of the pigmented areas of the rock art scenes, we can confirm that the P-ASK framework proposed in this study provides very satisfactory results for the camera characterization. Figure 17. Δ E a b * mapping image (96-24 patches). 4. Discussion The generic aim of clustering applications is the natural grouping of specimens that share similar properties within a data set. In this study, we established the P-ASK framework which leverages the K-means technique for automatic training in the selection of a simplified set of color patches, and properly capture the dominant color characteristics of rock art scenes. Since the K-means algorithm is sensitive to the initial approximations given by the user [ 56 , 57 ], a set of preliminary experiments were conducted to set proper values for the number of clusters, the working algorithm and the initialization method. According to our results, the suggested parameters to run K-means were appropriate, and the algorithm implemented in the KMeans function of the Scikit-learn module proved to be a valid option for clustering operations as well. However, the K-means algorithm is slow for large data sets, particularly those based on digital images. In our paper, we suggest an alternative approach to extract the dominant colors which consists of using a reduced number of image samples clipped from the full image. By using image sampling, the running time was considerably reduced. For instance, run time decreased from 18.2 min to 4.9 min when using the Fujifilm IS PRO image for K = 24 clusters (cf. Table 3 with Table 1 in Section 3 ). The experiment conducted to identify the effect of randomness on the K-means clustering technique showed that the dominant color set returned from multiple runs was highly consistent [ 48 ]. In this regard, Figure 8 shows Δ E a b * color differences less than 1 CIELAB unit for the dominant colors obtained in 100 realizations, with similar negligible results in units of pixel RGB digital values. Moreover, the capability of P-ASK to identify the dominant colors into the ColorChecker offers very good results (see Table 4 and Figure 13 ), and allowed extracting a set of color chips from the chart to create a reduced training data set for camera characterization in a simple and fast way. By using this framework, we proved that the characterization approach based on the KDP training data gives accurate results which parallel those obtained in common practice, that is, using the whole set of color chips for model learning. Although previous studies recommend the use of large training data sets for camera characterization [ 22 , 41 , 73 , 74 ], the results obtained with the P-ASK framework show that it is possible to use a reduced number of training samples without loss of accuracy in colorimetric terms. It is worth noting that the CIE XYZ residuals ( Table 5 ) and the Δ E a b * color differences ( Table 6 ) obtained using the KPD data set are in fact slightly better. In view of the outcome images, it is evident that sRGB characterized images were satisfactorily obtained ( Figure 16 ), which is confirmed by the color difference map image where differences under 2 CIELAB units prevail over the whole image, particularly in the area of interest for archaeological applications ( Figure 17 ). As an aside, we would like to note that the specific imaging hardware, i.e., the digital camera used for graphical documentation, is a key point that must be considered prior to characterization. We know from previous research that built-in imaging sensors have a considerable impact on the final outcome. In this sense, we presented the results of the Fujifilm IS PRO camera which is known to work very well, not only in the characterization procedure [ 25 ], but also in terms of image noise [ 75 ]. 5. Conclusions In this paper, we propose a new P-ASK framework for consistent and accurate archaeological color documentation. The graphic and numeric results obtained after the characterization of rock art images are highly encouraging and confirms that P-ASK is a suitable technique to use in the context of the image-based camera characterization procedure. Our characterization approach based on a reduced set of dominant colors offers several advantages. On the one hand, it implies the reduction of the training set size, and thereby, the computing time required for model training is less than in the regular approach with all color chips. On the other hand, there is an improvement derived from the use of a reduced number of specific color patches, which adjust better to the chromatic range of the scene, and yields better results in the output characterized images. We consider this point as a major contribution in this paper. The tests conducted in our study confirmed that P-ASK is robust to random issues in the K-means stage and works very well with a specific sampling of the elements of interest in the rock art scene. This point leads us to envision future research lines focused on developing specific color charts for archaeological applications, and even for specific cultural heritage objects, monuments and sites. In summary, the results achieved in this study show that P-ASK is a suitable tool for the proper and rigorous use of color camera characterization in archaeological documentation, and can be integrated into the regular documentation workflow with minimum effort and resources. Author Contributions Conceptualization, A.M.-T.; Formal analysis, A.M.-T.; Funding acquisition, J.L.L.; Methodology, A.M.-T.; Software, A.M.-T. and Á.M.-M.; Supervision, Á.M.-M., J.L.L. and S.W.; Writing—original draft, A.M.-T.; Writing—review & editing, Á.M.-M., J.L.L. and S.W. All authors have read and agreed to the published version of the manuscript. Funding This research is partly funded by the Research and Development Aid Program PAID-01-16 of the Universitat Politècnica de València, through FPI-UPV-2016 Sub 1 grant. Acknowledgments The authors would like to thank the Direcció General de Cultura i Patrimoni, Conselleria d’Educació, Insvestigació, Cultura i Esport de la Generalitat Valenciana, their authorization to carry out the 3D documentation in the Cova dels Cavalls site in Tírig (Castelló, Spain). The authors are also very grateful for the support of the staff at Colour Research Group from the University of Leeds (UK), who kindly allowed the use of their facilities and colorimetric instrumental during a research fellowship of the corresponding author. Conflicts of Interest The authors declare no conflict of interest. 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Randomness assessment: ( a ) Δ E a b * color differences ( b ) Standard deviation error. Figure 9. Graphic results for the SS method: ( a ) Patches identified; ( b ) Processing time. Figure 10. DigiEye imaging system: ( a ) DigiEye system (without camera integrated) ( b ) Nikon D40 test image (D65 illuminant). Figure 11. P-ASK framework adapted to the DigiEye D65 image acquired with the Nikon D40. Figure 12. Graphic description of the pigment sample (P1): ( a ) clipped detail image, ( b ) K-means centroids, ( c ) Dominant color palette, ( d ) Dominant color frequency histogram. Figure 13. Color patches detected: ( a ) Support samples; ( b ) Pigment samples; ( c ) Support-pigment; ( d ) KDP training samples. Figure 14. LOOCV CIE XYZ Residuals histogram: ( a ) FCC (24 patches) ( b ) KDP (24 patches). Figure 15. Δ E a b * color differences: ( a ) FCC (24 patches); ( b ) KDP (24 patches). Figure 16. Output sRGB characterized images: ( a ) FCC training data (96 patches) ( b ) KDP training data (24 patches). Figure 17. Δ E a b * mapping image (96-24 patches). Table 1. Evaluation of K-means running time with different values of K. K Nikon D40 Sigma SD15 Fujifilm IS PRO Mean Time (min) Time (min) Time (min) Time (min) 10 8 5.2 7.6 6.9 20 17.1 16.7 15.9 16.6 24 19.3 18.4 16.9 18.2 30 27.4 25.5 24.2 25.7 40 31.8 30.8 30.6 31.1 50 45.1 37.9 38.1 40.4 60 49.5 49.2 53.5 50.7 80 67.8 60.2 69.1 65.7 100 87.9 76.0 100.3 88.1 Table 2. Comparison of four methods to identify dominant color patches. Camera NN RC JS SS NikonD40 18 5 13 17 Sigma SD15 21 12 13 16 Fujifilm IS PRO 15 12 13 15 Average 18 10 13 16 Table 3. Assessment of the SS method for the identification of dominant patches. Nikon D40 (DSC0001) Sigma SD15 (SDIM0196) Fujifilm IS PRO (DIR0264) K Patches Time Patches Time Patches Time 10 14 3.6 15 1.7 15 2 20 15 8 16 3.3 15 4.3 24 17 9.7 16 4 15 4.9 30 17 9.3 16 5 15 5.8 40 18 12.8 17 6 16 6.5 50 19 14.9 17 7.2 17 9 60 20 16.8 17 8.7 17 10.5 80 21 23.1 17 11.6 17 13.4 100 21 26.3 17 13.2 17 16 Table 4. Color Patches detected from the Fujifilm IS PRO samples. Sample Dim Patches id S1 995 × 515 4 F7, J8, J9, K2 S2 620 × 950 4 F7, H7, J9, K2 S3 453 × 691 6 F7, G7, I7, J8, J9, K2 S4 725 × 321 2 F7, J9 S5 185 × 1406 9 E7, F7, G7, H7, H8, I8, J8, J9, K2 Support 9 E7, F7, G7, H7, H8, I8, J8, J9, K2 P1 222 × 204 12 D8, F2, F7, F8, G7, G8, H7, H8, I7, I8, J8, K2 P2 252 × 126 11 D8, E7, F2, F7, F8, G8, H7, H8, J7, J8, K2 P3 177 × 88 10 D8, F2, F7, F8, G7, G8, H7, H8, I8, K2 P4 192 × 108 12 D8, F2, F7, F8, G7, G8, H7, H8, I7, J7, J8, K2 P5 105 × 92 12 D8, F2, F7, F8, G7, G8, H7, H8, I7, J7, J8, K2 P6 161 × 63 12 D8, F2, F7, F8, G7, G8, H7, H8, I7, I8, J8, K2 P7 84 × 51 12 D8, E7, F2, F7, F8, G7, G8, H7, H8, J7, J8, K2 Pigment 14 D8, E7, F2, F7, F8, G7, G8, H7, H8, I7, I8, J7, J8, K2 Support & Pigment 15 D8, E7, F2, F7, F8, G7, G8, H7, H8, I7, I8, J7, J8, J9, K2 Table 5. CIE XYZ Residuals of the FCC and KDP models. FCC Model KDP Model (96) (24) (24) Stats CIE X CIE Y CIE Z CIE X CIE Y CIE Z CIE X CIE Y CIE Z Max 3.501 2.505 1.184 1.922 1.605 1.183 2.670 2.434 1.347 Min −4.985 −3.852 −1.612 −4.985 −3.852 −1.587 −3.523 −2.591 −1.090 Mean 0.017 0.010 0.006 −0.428 −0.359 −0.156 0.013 0.008 0.003 Std 1.387 1.050 0.654 1.640 1.245 0.704 1.252 0.954 0.553 Table 6. Δ E a b * color differences in the FCC and KDP characterization experiments. FCC Model KDP Model (96) (24) (24) Max 9.413 4.192 3.755 Min 0.322 0.497 0.179 Mean 2.107 1.868 1.518 Std 1.472 1.055 0.954 MDPI and ACS Style Molada-Tebar, A.; Marqués-Mateu, Á.; Lerma, J.L.; Westland, S. Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation. Remote Sens. 2020, 12, 520. https://doi.org/10.3390/rs12030520 AMA Style Molada-Tebar A, Marqués-Mateu Á, Lerma JL, Westland S. Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation. Remote Sensing. 2020; 12(3):520. https://doi.org/10.3390/rs12030520 Chicago/Turabian Style Molada-Tebar, Adolfo, Ángel Marqués-Mateu, José Luis Lerma, and Stephen Westland. 2020. "Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation" Remote Sensing12, no. 3: 520. https://doi.org/10.3390/rs12030520 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Article Metrics Article Access Statistics Multiple requests from the same IP address are counted as one view. MDPI and ACS Style Molada-Tebar A, Marqués-Mateu Á, Lerma JL, Westland S. Dominant Color Extraction with K-Means for Camera Characterization in Cultural Heritage Documentation. Remote Sensing. 2020; 12(3):520. https://doi.org/10.3390/rs12030520	https://www.mdpi.com/2072-4292/12/3/520/xml
Summary Charlie Parker, Called "Bird," Was a World-Famous Alto Saxophonist and Jazz Composer - DocsLib Landmarks Preservation Commission May 18, 1999, Designation List 304 LP-2032 CHARLIE PARKER RESIDENCE, 151 Avenue B (aka Charlie Parker Place), Borough Landmarks Preservation Commission May 18, 1999, Designation List 304 LP-2032 CHARLIE PARKER RESIDENCE, 151Avenue B(akaCharlie ParkerPlace), Borough ofManhattan. Built circa 1849, architect undetermined. Landmark Site: Borough of Manhattan Tax Map Block 392, Lot 5 On December 15, 1998, the Landmarks Preservation Commission held a public hearing on the proposed designation as a Landmark of the Charlie Parker Residence and the proposed designation of the related Landmark Site (Item No. 5). The hearing had been duly advertised in accordance with the provisions of law. The six speakers who spoke in favor of designation were representatives of the owner, the Stuyvesant Park Neighborhood Association, the Historic Districts Council, the Municipal Art Society, andjazzhistorianPhil Schaapwho played a brief taped interview with drummerMax Roach. The Commission has received numerous statements in support of this designation, including letters from Community Board Three Manhattan, (former) Borough President of Manhattan Ruth W. Messinger, City Councilmembers Margarita Lopez and Antonio Pagan, the East Village Parks Conservancy,New YorkLandmarks Conservancy, as well as dozens of local residents and jazz aficionados. There were no speakers or letters in opposition. Summary Charlie Parker, called "Bird," was a world-famous alto saxophonist and jazz composer. From late 1950 through October 1954 he and his common-law wife Chan Richardson occupied the ground floor of No. 151 Avenue B, located rnidblock between East 9th and East 10th Streets, oppositeTompkins Square Park. Two children, a boy and girl, were born to the couple during their residency here. Parker first arrived inNew York Cityin 1938 or 1939, and soon established himself as one of jazz's most gifted and influential performers. He moved into the apartment at the height of his career, having achieved considerable success and renown as the co-founder ofbebop, the modem jazz style that he and trumpeter Dizzy Gillispie created in New York City during the mid-1940s. While living on Avenue B, Parker enjoyed international fame; recording with small and large ensembles, as well as with Latin big bands and string sections. The Parker residence is also a rare example of a rowhouse designed in the Gothic Revival style. Built circa 1849, the most significant architectural element is the well preserved pointed arch entranceway with clustered colonettes that is surmounted by a prominent horizontal hood mold. Original details include the double wood doors, a trefoil relief beneath the projecting box cornice, as well as the slender hood moldings above most windows. Now painted, thebrownstonefacade was restored in 1994-95. 1 DESCRIPTION AND ANALYSIS Charlie Parker (1920-1955) Parker met Chan Richardson (born Beverly Few musicians have left as lasting a mark on Dolores Berg) in 1943. Raised in Yonkers, New twentieth century music as the alto saxophonist York, Richardson was a dancer and jazz enthusiast. Charlie Parker. Nicknamed "Bird" or "Yardbird, " When they first met, she was living on West 52nd Parker is credited with creating a new style of jazz in Street, beside the dozen or more jazz clubs clustered New York City during the mid-1940s, known as bop between Fifth and Sixth Avenues. Prior to settling 2 ( or bebop). Born in Kansas City, Kansas, Parker down with Richardson in late 1949, Parker was came to New York in late 1938 or 1939. Since the married three times, to Rebecca Ruffing (1936), late 1920s, Manhattan had been the nation's jazz Geraldine Scott (1941), and Doris Sydnor (1948). In center, attracting such celebrated musicians as Louis New York be moved frequently, rarely remaining at Armstrong andDuke Ellingtonto its nightclubs and a single address for more than a few months. He lived recording studios. After several months working as a at severalHarlemlocations, including 411 Manhattan dishwasher, Parker secured a job performing popular Avenue, on Amsterdam Avenue and 149th Street, in tunes at the "dime-a-dance" Parisien Ballroom, on the Dewey Hotel on West 117th Street, as well as inBroadwaynear 48th Street. The young saxophonist the Marden Hotel on West 44th Street, near Times soon gained notice from the jazz community, as a Square. As an ascendant jazz artist, these addresses featured soloist in Jay McShann's Big Band, and were convenient, close to the venues where he during frequent after-hours jam sessions at Harlem frequently performed, such as Minton's Playhouse nightclubs, such as Clark Monroe's Uptown House (West 118th Street), Small's Paradise (Adam Clayton and Dan Wall's Chili House. Powell Boulevard and 135th Street) and the Royal As a member of theEarl HinesBig Band during Roost (Broadway and47th Street). 1943, Parker met trumpeter John Birks "Dizzy" The sole exception to this pattern occurred during Gillespie. A musical bond quickly formed, described his relationship with Richardson. Although never by musicologist Carl Woideck as "the most vital legally married, Richardson took Parker's name. partnership in jazz of the time. "3 Their early Biographer and jazz criticGary Giddinswrote that she recordings challenged the status quo; unlike swing, was: which developed a popular following during the 1930s, bebop's character was "jagged, nervous, . . . the only one of his wives, licensed or esoteric, and technically brilliant. "4 Though now common-law, who called him Bird instead of widely admired, initially, such recordings as Charlie, Chan was a fan, who also recognized 6 "Groovin' High," "Salt Peanuts," and "Koko" Bird's desire for bourgeois stolidity. received negative reviews. While some critics contended that their records "did not add up to good Parker and theLower East Sidejazz, "5 these performances defined Parker's sound, Between 1950 and 1954, Parker and Richardson anointing him leader of a new generation of lived on the Lower East Side, far from the nightclubs musicians. and recording studios that fueled his career, as well as In late 1945 Parker traveled to Los Angeles, the drug dealers in Spanish Harlem that profited from where be suffered a nervous breakdown. He spent six his heroin addiction. In May 1950, Richardson and months at California's Camarillo State Hospital, her daughter Kim moved into his apartment at No. recuperating and regaining his general health. Upon 422 East 11th Street, betweenFirst Avenueand his return to New York in April 1947, he formed theAvenue A(demolished). so-called "classicquintet" featuring drummer Max Toward the end of the year, they relocated to No. Roach and trumpeterMiles Davis. During his absence 151 Avenue B, leasing the ground floor in a century old rowhouse that had recently been converted to from New York, bebop's popularity grew and 7 Parker's quintet benefitted greatly, performing at apartments. Richardson recalled in her 1993 more prestigious nightclubs and with higher pay. Live autobigraphy: radio broadcasts from such locations as the Royal Our apartment was in the Ukrainian section Roost and other midtown venues spread Parker's of the Lower East Side . . . It was an area fame, as did the numerous recordingsNorman Granzfull of poverty, peopled by Hasidic Jews with produced for the Mercury label between 1948 and side locks and gypsies, and was a melting pot 1954. for refugees. Our building was across from Thompkins (sic) Square Park and each of the 2 five families living in it occupied an entire topped Metronome and Downbeat magazine's annual floor. We occupied the ground floor, which reader's poll. He recorded prolifically, with small and had a separate entrance and opened onto a large groups, as well as with Latin big bands and large courtyard in back for the children and string sections. Some of these performances became all the animals we would eventually legendary, including "The Quintet of the Year," accumulate. We redecorated the apartment. recorded in Toronto'sMassey Hallon May 15, 1953. We bought blond wood furniture, put up Featuring drummer Max Roach, pianistBud Powell, Steinberg birdcage wallpaper from Sloans and bassistCharles Mingusand Gillispie, the recording, painted the living room charcoal grey. 8 reissued as "The Greatest Jazz Concert Ever, " was Parker and Richardson had two children while living inducted into the Grammy Hall of Fame in 1995. here: a daughter, Pree, born in 1951, and a son, Furthermore, two Parker recordings became popular Baird, born in 1952. She described this period of hits:"My Little Suede Shoes" in 1951, and "Autumn Parker's life as one of "happy maturity," observing: in New York, " recorded with a 40-pieceorchestrain 1952. He had entered this phase of domestic Parker's desire for a stable family life, however, stability and his children brought much joy to proved elusive. Though never jailed for drug him. He dug Sundays best of all. I would possession, his "cabaret card, " which permitted him cook a roast and my mother, brother Jimmy, to work in New York State nightclubs, was revoked and Aunt Janet would come for Sunday in July 1951. With a wife and three children to dinner. It was all very middle class, except support, Parker spent much of the next two years on for our table which we had made in the form the road. During these lengthy concert tours he drank of a G, or treble, clef. The indentation was incessantly, suffered bleeding ulcers, and depression. 9 the perfect spot for a high chair. In a final blow, his· daughter Pree, less than three The neighborhood's Eastern European flavor brought years old, died of congenital heart failure in March 12 Parker considerable pleasure. He appreciated its lack 1954. Devastated by the loss, Parker attempted of "hype"10 and frequented restaurants, bars, and even suicide twice. In October 1954 they moved to New nightclubs in the area. Parker told drummer Ed Hope, Pennsylvania, and by Christmas he and 13 Shaughnessy: Richardson had separated. Parker made his last commercial recordings in . . . you've got to come up to this Rumanian December 1954, and three months later, on March restaurant with me. They have a fantastic 5th, his final public appearance -- at Birdland. He folk group with authentic stringed instruments died nearly penniless on March 12, 1955 in a fan's and percussion, and you know something, suite at the Stanhope Hotel, located at Fifth A venue they swing more than we do! and 80th Street. The funeral took place nine days later Though the apartment never became a musician's at the Abyssinian Baptist Church (a designated New hangout, colleagues did occasionally visit, including York City Landmark), on West 138th Street. Parker pianistJoe Albanyand saxophonist Al Cohn. Cohn is buried in Kansas City. remembered: "Bird" Remembered They had a very nice place. After a while, In the years following his death, Parker achieved he told Chan we were going out for a few legendary status. Tributes to the man and his music drinks. It was a Ukrainian neighborhood and have remained constant, from the graffiti "Bird we went to three or four different bars. All Lives!" scrawled on the walls of the subway and in the Ukrainians, working-class guys, knewGreenwich Villageduring the late1950s, to the jazz him as Charlie. I don't think they knew he inflected writings of the Beat poets, especially Jack was a musician, but it was obvious they liked Kerouac andAllen Ginsberg. 14 Many of his finest him and were glad to see him. I saw a studio recordings and concert performances remain different side of him, he was like a middle available on compact disc and he has become the 11 class guy with middle-class values. subject of numerous books, dissertations, as well as While living on Avenue B, Parker enjoyed the Hollywood film, Bird, directed byClint Eastwoodinternational fame, touring theUnited States, Canada, (1988). In a 1996 analysis of Parker's music, Carl and Europe. The city' s largest jazz venue "Birdland" Woideck described him as: (at 1678 Broadway, near52nd Street) was named in . . . one of the most influential of all jazz his honor in December 1949, and he frequently musicians, regardless of era. His position in 3 jazz is analogous toLouis Armstrong's in that residence. Designed in the Gothic Revival style, its both musicians advanced the music that they monumental stone facade may have served as had inherited . . . inviting all jazz inspiration for the undetermined architect who instrumentalists and composers of any era to designed No. 151 Avenue B -- the subject of this reevaluate every aspect of their art. 15 report. Many brick and brownstone residences were Bird.flight, a program devoted exclusively to Parker's constructed along the east side of the park during this music has had a permanent place on New York City period, including Nos. 149, 151, and 153 Avenue B. radio since April 1981. Hosted by jazz historian Phil Built by 1849, No. 151 is the best-preserved, airs retaining its high stoop, box cornice, and considerable Schaap, it each weekday morning on WKCR-FM, 18 New York, the radio station ofColumbia University. 16 Gothic Revival style detail. J_udith Rhodes, a producer of jazz concerts, acqmred the Parker Residence in 1979, in part The American Gothic Revival because of its unique association with the musician. In .1:he Charlie Parker Residence is a fairly 1992, Avenue B, between 7th and 10th Streets, was t~ditional rowhouse, three stories tall and three bays renamed "Charlie Parker Place," and Tompkins wide. What sets the house apart from most mid Square Park has become the site of an annual August nineteenth century dwellings in New York City is the co_ncert celebrating his birthday. First held in 1993, use of the Gothic Revival style. Frequently adopted by this popular free event features performances by church designers, this style rarely appears in private musicians who played with Parker as well as those residences. Popularized in England, by the mid-1830s influenced by his music. In 1994, the "Charlie Parker this picturesque style had crossed the Atlantic Residence" was listed on the National Register of !nspiri_ng the _d~ign of numerous houses of worship: Historic Places. 17 mcludmg Tnmty Church (Richard Upjohn, 1839- A performer of impressive virtuosity, Parker's 1846, a designated New York City Landmark) and music continues to attract a devoted international Holy Trinity Church/now St. Anne and Holy Trinity audience. Despite a tragically-shortened career, "Bird" Episcopal Church (Minard Lafever, 1844-1847, remains one of the twentieth century's most-celebrated located in the Brooklyn Heights Historic District), and musicians. Anshe Chesed Synagogue (Alexander Saeltzer, 1849- 1850, a designated New York Landmark) on the Tompkins Square Lower East Side. The Charlie Parker Residence is located opposite Andrew Jackson Downing (1815-1852) played an Tompkins Square Park, on the east side of A venue B critical role in advancing the Gothic Revival style in between 9th and 10th Streets. Originally part of Pete~ the United States. In Cottage Residences (1842) and Stuyvesant's farm, during the eighteenth century the The Architecture of Country Houses (1850) he area was known as Stuyvesant Meadows. The presented numerous designs, illustrated with detailed Commissioner's Plan of 1811, which established the floorplans and elevations. These pattern books proved 19 city's grid system, proposed a series of public parks, immensely popular and they remain in-print to this as well as a large public market, east of First A venue day. In a chapter titled "What a Country House or near what is now Tompkins Square. Initially called Villa Should Be" from The Architecture of Country Clinton Square, in 1833 the market square was Houses, he praised the Gothic style as "poetic 20 renamed for the former Governor of New York and inspiring, imaginative. " Downing's influence w~ Vice President of the United States, Daniel D. felt primarily in rural communities, where "high . Tompkins. A year later the grounds were leveled, gables wrought with tracery" were often incorporated 21 fenced, and planted with trees, and by 1847 gas into wood dwellings. While many examples of this lighting had been installed. type survive in the Hudson River Valley, especially Despite such civic improvements, by the late near Newburgh, New York, only a small number exist 1840s the square was used primarily for military in New York City, including the Alice Austen House exercises and recruitment by the prestigious Seventh (remodeled 1846, a designated New York City Regiment. Such activities may have discouraged real Landmark) and the W .S. Pendleton House (attributed estate development; whereas numerous fine residences to Charles Duggin, c. 1855, a designated New York stood near Washington and Union Squares, the blocks City Landmark), both on Staten Island. surrounding Tompkins Square had a less coherent Due to laws prohibiting wood construction in character. In 1848, St. Brigid's Roman Catholic urban areas, most nineteenth century dwellings in Church was built at the southeast comer of A venue B New York and Brooklyn were built of brick or and 8th Street, two blocks south of the future Parker brownstone. While the Federal and Greek Revival 4 style, as well as the Italianate style tended to 1955. While he liked the neighborhood, commenting dominate, a few surviving examples of the Gothic that it reminded him of England, he remained for only Revival style can be found in New York City three months. Fielding Dawson, who wrote a memoir landmark districts, including Greenwich Village, of Kline's life, described the apartment as "a beautifulGramercy Park, and most notably, Brooklyn Heights. top floor place on A venue B, too small, more for a Significant examples are located at Nos. 131 and 135 writer, and so he moved again. "24 From 1962 to Hicks Street, as well as a free-standing house at No. 1966, Agostini occupied an upper floor apartment. 36 Pierrepont Place, at Hicks Street. As is the case of During these years his work in cast-plaster and bronze the Charlie Parker Residence, evidence of the style was exhibited internationally and collected by tends to be limited to the entrance and window American art museums, such as the Museum of surrounds. Modem Art, the Metropolitan Museum of Art, and the Hirshhom Museum and Sculpture Garden. Subsequent History In 1866, Tompkins Square was declared a public Description parade ground. The neighborhood's Irish Catholic The Charlie Parker Residence is located on the population soon gave way to Germans who dominated east side of Avenue B, opposite Tompkins Square the area well into the twentieth century. Avenue B Park. Now painted off-white, No. 151 Avenue B is became an important north-south thoroughfare, served the block's only brownstone rowhouse to retain a by horsecars, and later, cable cars. Furthermore, stoop. The stoop (painted, with non-historic handrails beginning in 1853 I 0th Street offered commuters the at either side) ascends to a deeply-carved, pointed most direct route to the ferry piers serving Greenpoint arch doorway, consisting of paired colonettes that rise in Brooklyn. and intersect over the door. Above the pointed arch is After 1880 the house was sold to David Toal who a prominent horizontal hood molding. To the south, divided the building into two apartments. William just below where the hood molding ends, a small Arch, a bottle dealer, and his wife Annie, probably rectangular brass plaque identifies the year the occupied the ground floor, while Sarah Spenade, a building was constructed: "1849." The painted double widow, lived in the upper stories with her son and wood doors, one step up and set inside the arch, are two daughters, as well as a boarder, the physician original. Framed by wood colonettes, each door Joseph Vanderbergh. During the late nineteenth consists of five carved trefoil panels. The vertical century the northern section of the Lower East Side panels, at center, have been partially replaced by was known as Kleindeutschland, Little Germany, and glass. In addition, a metal mail slot has been inserted Dutchtown. Most residents were German immigrants, into the center of the horizontal panel in the north and a significant group of philanthropic institutions door. Centered above the doors is the building' s located closeby, such as the Children's Aid Society address, identified with non-historic individual metal Boys Lodging House at No. 127 Avenue B, numbers. There is a large, single plate of glass above Christadora House, a social service facility at No. 147 the doors, as well as a small non-historic lighting Avenue B (1928), and the Tompkins Square branch of fixture. the New York Public Library (McKim, Mead & There are two long windows beside the first floor White, 1904). entry, as well as three smaller ones across the second In 1906 the house was converted into the Bryson and third floors. All are the same width, one-over Day Nursery. Over the next decades, the area's one, and framed by thin hood moldings and sills. All population became increasingly diverse, attracting of the windows are non-historic, except on the first Italian, Eastern European, Russian, and later, Puerto floor, which are made of wood. Across the top of the Rican residents. In February 1945 the nursery school facade a painted box cornice is adorned with was purchased by Joseph Raskob. That spring the decorative moldings. The cornice's raised pattern building was divided into three apartments, with a set alternates between rectangles and squares. An inverted back penthouse on the roof.22 During Parker's molding extends down from the cornice, as well as a residency, Raskob sold the building to Christadora raised trefoil relief that is painted to match the facade. House, which owned it from 1952 to 1958. On the roof, visible from the street, an iron fence is After Parker moved in 1954, the building had two installed. The penthouse (or fourth level) is not other tenants of note: the painter Franz Kline (1910- visible from the sidewalk across A venue B. 1962) and the sculptor Peter Agostini (1913-1993).23 The ground level, where Parker lived, has two Kline, a celebrated member of the Abstract windows set at grade. Here the facade is slightly Expressionists, occupied the third floor briefly during raised, establishing a line below both the first story 5 and ground level windows. Vertical security gates, stairs, an iron fence extends along the sidewalk to the with three cross bars, shield the lower windows. edge of the property. Close to the stairs is a gate that Between the windows, a small, nearly square, brass opens and permits access to the ground floor. Parallel plaque is attached to the facade. It reads: 1950- to the fence is a second, taller fence that turns east at 1954/THE/CHARLIE PARKER/RESIDENCE/HAS a right angle to meet the facade. BEEN PLACED ON THE/NATIONAL REGISTER OF IDSTORIC PLACES/BY THE UNITED STATES DEPARTMENT OF THE INTERIOR." The ground floor apartment is reached by a step and through an iron gate set beneath the stairs. South of the door is a Report Prepared by stone placque, carved with the address "I 51." At the Matthew Postal sidewalk the stairs are framed by a pair of non Research Department historic fences that do not meet and intersect with the descending handrails on the first step. South of the N01ES 1. Since the establishment of New York City Landmarks Commission in 1965 a number of properties have been designated for their combined architectural and cultural significance. Examples include the Louis Armstrong House at 34-56 107th Street inQueens, theLangston HughesHouse at 20 East 127th Street in Manhattan, and the Edgar Allen Poe Cottage at 2640 Grand Concourse in the Bronx. 2. The term bebop is said to have its origins in musical sounds. Others have suggested that it was invented by Fats Waller. SeeRoss Russell, Bird Lives! The High Life and Times of Charlie (Yardbird) Parker (New York: Charterhouse, 1973), 136. 3. Carl Woideck, Charlie Parker: His Music and Life (Anne Arbor: The University of Michigan Press, 1996), 110. 4 . Bany Kernfeld, "Jazz," The Encyclopedia of New York City (New Haven: Yale University Press, 1995), 615. 5. Down Beat, April 22, 1946, cited by Russell, 197. 6. Gary Giddins, Celebrating Bird: The Triumph of Charlie Parker (Beech Tree Books, 1987), 109. 7. Russell, 302. 8. Chan Parker, My Life in E-Flat (Columbia, S.C.: University of South Carolina Press, 1993), 31. 9. Ibid., 34. 10. From George Reisner, Bird: The Legend of Charlie Parker (New York: Bonanza Books, 1962), 11. 11. Ibid., 109. 12. Pree Parker died March 6, 1954 in St. Vincents' Hospital, on Seventh Avenue near 12th Street. 13. Parker returned to the Lower East Side in January 1955, pawning hissaxophoneat Edelstein Brothers, 233 East14th Street. Photograph reproduced in Giddins, last page. 14. During 1953Jack Kerouaclived at 501 East 11th Street, about two blocks from Parker. See The Encyclopedia of New York City, 635. It should also be noted that in May 1997 Allen Ginsberg sent a letter supporting the designation of the Parker house, see Charlie Parker Residence files, Landmarks Preservation Commission. 15. Woideck, vii. 16. Phil Schaap began programming Parker's music on WKCR in August 1973. According to Schaap, "Birdflight" is the station's most listened-to program. Conversation with Schaap, April 1999. 6 17. National Register of Historic Places, Charlie Parker Residence, U.S. Department of the Interior, Heritage, Conservation and Recreation Services (form prepared by Jeffrey L. Baumoel, 1994). 18. This date is based on the interpretation of increased property values between 1848 and 1849. See National Register of Historic Places, Charlie Parker Residence, section 7, 3. 19. Cottage Residences (1842) went through four editions and The Architecture of Country Houses (1850), nine printings, equaling more than six thousand copies. See A. J. Downing, The Architecture of Country Houses (New York: Dover Publications, 1969), introduction by J. Stewart Johnson, vii. 20. Downing, The Architecture of Country Houses, 262. 21. Ibid., 274. 22. NYC, Department of Buildings, Block 392, lot 5, Permit No. AL.557-45, dated April 10, 1945. Cited by Baumoel in National Register of Historic Places report, Charlie Parker Residence, section 7, 3. 23. For a detailed chronology of Kline's life, see Harry F. Gaugh, The Vital Gesture: Franz Kline (Cincinnati A.rt Museum, 1985), especially p. 179. Information on Agostini was collected by Jeffrey L. Baumoel, a preservation consultant, who submitted a "Request for Evaluation" to the Landmarks Commission in September 1996. See Research files, New York Landmarks Commission. 24. Fielding Dawson, An Emotional Menwir of Franz Kline (New York: Pantheon Books, 1967), 27-28. 7 FINDINGS AND DESIGNATION On the basis of a careful consideration of the history, architecture and other features of this building, the Landmarks Preservation Commission finds that the Charlie Parker Residence has a special character, special historical and aesthetic interest and value as part of the development, heritage, and cultural characteristics of New York City, New York State, and the nation. The Commission further finds that, among its important qualities, the Charlie Parker Residence was occupied by the world-famous alto saxophonist Charlie Parker from 1950 to 1954 at the height of his career; that Parker was the co-founder of the modem jazz style bebop; that he moved into the ground floor apartment of the three-story rowhouse; that the brownstone-fronted building (now painted), built circa 1849, is a rare surviving example of a Gothic Revival style residence; that among its significant features are the pointed-arch entranceway with horizontal hood molding and the raised trefoil relief below the box cornice; and that as Parker's home for four years, the building remains the most significant site in New York City associated with Parker and the only site in New York City with which he was associated for an extended time. Accordingly, pursuant to the provisions of Chapter 74, Section 3020 of the Charter of the City of New York and Chapter 3 of Title 25 of the Administrative Code of the City of New York, the Landmarks Preservation Commission designates as a Landmark the Charlie Parker Residence, 151 Avenue B, Borough of Manhattan, and designates Borough of Manhattan Tax Map 392, Lot 5, as its Landmark Site. 8	https://docslib.org/doc/12456561/summary-charlie-parker-called-bird-was-a-world-famous-alto-saxophonist-and-jazz-composer
Continued. \| Download Scientific Diagram Download scientific diagram \| Continued. from publication: Relaxin and Castration in Male Mice Protect from, but Testosterone Exacerbates, Age-Related Cardiac and Renal Fibrosis, Whereas Estrogens Are an Independent Determinant of Organ Size \| This study determined the effects of castration and hormone replacement therapy on the age-related cardiac and renal pathology of male relaxin gene-knockout (RlnKO) and age-matched wild-type (RlnWT) mice and that of aged male aromatase knockout (ArKO) mice, which lack... \| Relaxin, Castration and Mice \| ResearchGate, the professional network for scientists. Continued. Source publication Relaxin and Castration in Male Mice Protect from, but Testosterone Exacerbates, Age-Related Cardiac and Renal Fibrosis, Whereas Estrogens Are an Independent Determinant of Organ Size Article Full-text available Chongxin Zhao Belinda Wigg This study determined the effects of castration and hormone replacement therapy on the age-related cardiac and renal pathology of male relaxin gene-knockout (RlnKO) and age-matched wild-type (RlnWT) mice and that of aged male aromatase knockout (ArKO) mice, which lack estrogens and have 5-10 times the androgen levels of male wild-type mice. One-mon... Contexts in source publication Context 1 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... Context 2 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... Context 3 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... Context 4 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... Context 5 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... View in full-text Context 6 ... BW, organ weights, and organ weight to body weight ratios of sham-operated, castrated, and TT- or ET- gonadectomized male mice are presented in Table 1. Twelve-month-old sham-operated (control) RlnKO mice had significantly larger LVs and kidneys (by 10 and 14%, respectively) in addition to LV to BW and kidney to BW ratios (by 14 and 21%, respectively), compared with age- matched RlnWT animals (all P Ͻ 0.05 vs. 12 month old control RlnWT mice with testes intact; Table 1). Castration of male RlnWT and RlnKO mice (at 1 month of age) led to a 17–22% reduction in their BW and significantly smaller hearts (by ϳ 30%), LV (by 26 –27%), and kidneys (by 35– 40%) by 12 months (all P Ͻ 0.01 vs. age matched control mice with intact testes). When corrected for BW, the kidney weights of 12-month-old castrated male RlnWT mice and heart and kidneys weights of age-matched castrated RlnKO mice were also smaller than their respective control counterparts (with testes intact). TT of castrated RlnWT and RlnKO mice normalized the gonadectomy-in- duced reduction in BW, organ weights, and organ weight to BW ratio to the values seen in respective control animals (all P Ͻ 0.05 vs. measurements from respective castrated mice; Table 1). Serum levels of testosterone in castrated ϩ TT mice were 412 Ϯ 172 pg/ml, but were below the detectable level of the testosterone RIA in castrated animals (n ϭ 5 mice/ group). The ET of castrated RlnWT mice also normalized the gonadectomy-in- duced reduction in BW, heart and kidney weights, and heart weight to BW and kidney weight to BW ratios (all P Ͻ 0.05 vs. measurements from respective castrated mice) and significantly increased LV weight and LV to BW ratio to levels above that measured in control RlnWT animals (both P Ͻ 0.05 control RlnWT mice; Table 1). When administered to castrated RlnKO mice, however, ET did not have any marked effects on BW and only partially normalized absolute heart, LV, and kidney weights, such that the weights of these organs in ET-castrated mice were higher than that measured in castrated mice but lower than that measured in TT-gonadectomized animals (Table 1). When corrected for BW, ET of castrated RlnKO mice ap- peared to normalize heart, LV, and kidney to BW ratio to that measured in control male RlnKO mice. Despite 12-month-old male RlnKO mice having larger kidney weights and kidney weight to BW ratios compared with age-matched RlnWT animals (Table 1), no significant differences in glomerular volume (Fig. 1, A and B) or density (Fig. 1, D and E) were measured between control male RlnWT and RlnKO mice. Consistent with the smaller kidneys seen in castrated animals, however, both gonadectomized RlnWT and RlnKO mice had smaller glomeruli (Fig. 1, A and B) and an increased glomerular density (Fig. 1, D and E). Conversely, both TT and ET restored glomerular volume (Fig. 1, A and B) and glomerular density (Fig. 1, D and E) in these animals in the same manner that they restored the castration-induced reduction in kidney weight and kidney weight to BW ratio (Table 1). Interestingly, no differences in glomerular volume (Fig. 1C) or glomerular density (Fig. 1F) were measured between intact 12-month-old male ArWT and ArKO mice, the latter being estrogen deficient and containing 5–10 times the androgen levels measured in age-matched male ArWT mice. Biochemical analysis of LV and kidney collagen concentration (percentage collagen content to dry weight tis- mals ( P Ͻ 0.05 vs. respective values from TT-RlnWT mice). Conversely, ET had no marked effects on the castration-induced normalization of pSmad2, such that LV and renal pSmad2 levels in castrated ϩ ET-RlnWT and RlnKO mice were significantly lower than that measured in castrated ϩ TT-animals ( P Ͻ 0.05 vs. respective levels measured in castrated ϩ TT-mice) (Fig. 3, A and B). Based on the strikingly similar pattern of changes in collagen concentration/staining (Fig. 2) and pSmad2 (Fig. 3) that were evident in the LV and kidney of relaxin-de- ficient, castrated, and TT- or ET-mice, the effects of ... Citations ... In this regard, Hewitson and his team reported that androgen deprivation by castration has imparted protection to the kidney from age-related fibrosis, where androgen replacement aggravated extracellular matrix accumulation. This is consistent with our results [47] . ... Androgen receptor blockade by flutamide down-regulates renal fibrosis, inflammation, and apoptosis pathways in male rats ... Endogenous relaxin deficiency in mice has been found to be associated with impaired development of several reproductive organs, leading to infertility in males [114], and an agerelated progression of fibrosis in several non-reproductive organs including the lung, heart, kidneys, and skin [115]. This led to renal cortical thickening and impaired kidney function in male mice [116,117], which was attributed to the higher levels of testosterone found in males in contrast with the protective effects of estrogen in females [118] . Based on these findings, which identified endogenous relaxin as a naturally-occurring regulator of collagen turnover, a recombinant form of human relaxin-2 (also known as serelaxin; which is also referred to as RLX) was developed to evaluate the anti-fibrotic potential of RLX, which was found to be bioactive in several other species including rodents [119]. ... Enhancing the Therapeutic Potential of Mesenchymal Stromal Cell-Based Therapies with an Anti-Fibrotic Agent for the Treatment of Chronic Kidney Disease Full-text available May 2022 Chronic kidney disease (CKD) affects 1 in 10 members of the general population, placing these patients at an increasingly high risk of kidney failure. Despite the significant burden of CKD on various healthcare systems, there are no effective cures that reverse or even halt its progression. In recent years, human bone-marrow-derived mesenchymal stromal cells (BM-MSCs) have been recognised as a novel therapy for CKDs, owing to their well-established immunomodulatory and tissue-reparative properties in preclinical settings, and their promising safety profile that has been demonstrated in patients with CKDs from several clinical trials. However, renal fibrosis (scarring), a hallmark of CKD, has been shown to impair the viability and functionality of BM-MSCs post-transplantation. This has suggested that BM-MSCs might require a pre-treatment or adjunct therapy that can enhance the viability and therapeutic efficacy of these stromal cells in chronic disease settings. To address this, recent studies that have combined BM-MSCs with the anti-fibrotic drug serelaxin (RLX), have demonstrated the enhanced therapeutic potential of this combination therapy in normotensive and hypertensive preclinical models of CKD. In this review, a critical appraisal of the preclinical data available on the anti-fibrotic and renoprotective actions of BM-MSCs or RLX alone and when combined, as a treatment option for normotensive vs. hypertensive CKD, is discussed. ... Testosterone and estradiol could affect different aspects of kidney physiology and health. Cellular processes such as fibrosis, collagen synthesis, inflammation, and apoptosis are differentially regulated by testosterone and estradiol, with beneficial effects attributed to estradiol (11) (12)(13)(14)(15)(16). Moreover, testosterone and estradiol could also affect kidney hemodynamic function, where testosterone has been shown to induce vasodilatory effects that may increase perfusion (27). ... Kidney hemodynamic function in men and postmenopausal women with type 2 diabetes and preserved kidney function Background Progression of kidney disease may differ between sexes in type 2 diabetes (T2D), with studies reporting slower decline in women. Glomerular hyperfiltration is a key factor driving kidney function decline. The current study aims to investigate the differences in kidney hemodynamic function between men and women with T2D. Methods A cross-sectional analysis of pooled data from three studies comparing kidney hemodynamic function between men and postmenopausal women with T2D without overt nephropathy. The outcome measures were GFR (inulin clearance), effective renal plasma flow (ERPF; para-aminohippurate clearance), filtration fraction (GFR/ERPF) and renal vascular resistance (RVR; mean arterial pressure/RBF). Glomerular hydraulic pressure (PGLO), afferent and efferent vascular resistance were estimated by Gomez-formulae. Sex differences were assessed with linear regression models adjusted for systolic blood pressure, glucose, use of renin-angiotensin-system blockers and body mass index. Results 101 men (age: 63[58-68] years; BMI 31.5±3.9 kg/m2; GFR 111±18 mL/min; HbA1c 7.4±0.7%) and 27 women (age: 66[62-69] years; BMI 30.9±4.5 kg/m2; GFR 97±11 mL/min; HbA1c 7.1±0.5%) were included. GFR was higher in men versus women (11.0 mL/min; [95%CI; 3.6;18.4]). Although statistically non-significant, PGLO trended higher in men (1.9 mmHg [95%CI; -0.1;4.0], while RVR (-0.012 mmHg/L/min [95%CI; -0.022;-0.002]) and afferent vascular resistance were lower (-361 dyne/sec/cm5 [95%CI;-801;78]). Conclusions In adults without overt nephropathy, GFR was higher in men compared to women. PGLO also trended to be higher in men. Both findings are possibly related to afferent vasodilation and suggest greater prevalence of hyperfiltration. This could contribute to accelerated GFR loss over time in men with T2D. ... Similarly, castration was found to be protective against fibrosis in male mice in the model, with testosterone replacement ultimately reversing this effect. Similar observations have been made in models of cardiac, kidney, and bladder fibrosis, indicating that sex alters tissue fibrosis in experimental animals (7) (8) (9). ... The Homeodomain Transcription Factor NKX3.1 Modulates Bladder Outlet Obstruction Induced Fibrosis in Mice Full-text available Mehul S. Patel Diana K. Bowen Nicholas M. Tassone Andrew D. Gould Robert W. Dettman Fibrosis is an irreversible remodeling process characterized by the deposition of collagen in the extracellular matrix of various organs through a variety of pathologies in children, leading to the stiffening of healthy tissues and organ dysfunction. Despite the prevalence of fibrotic disease in children, large gaps exist in our understanding of the mechanisms that lead to fibrosis, and there are currently no therapies to treat or reverse it. We previously observed that castration significantly reduces fibrosis in the bladders of male mice that have been partially obstructed. Here, we investigated if the expression of androgen response genes were altered in mouse bladders after partial bladder outlet obstruction (PO). Using a QPCR microarray and QRTPCR we found that PO was sufficient to increase expression of the androgen response gene Nkx3.1. Consistent with this was an increase in the expression of Nkx3.1 protein. Immunofluorescent antibody localization demonstrated nuclear Nkx3.1 in most bladder cells after PO. We tested if genetic deletion of Nkx3.1 alters remodeling of the bladder wall after PO. After PO, Nkx3.1KO/KO bladders underwent remodeling, demonstrating smaller bladder area, thickness, and bladder: body weight ratios than obstructed, wild type controls. Remarkably, Nkx3.1KO/KO specifically affected histological parameters of fibrosis, including reduced collagen to muscle ratio. Loss of Nkx3.1 altered collagen and smooth muscle cytoskeletal gene expression following PO which supported our histologic findings. Together these findings indicated that after PO, Nkx3.1 expression is induced in the bladder and that it mediates important pathways that lead to tissue fibrosis. As Nkx3.1 is an androgen response gene, our data suggest a possible mechanism by which fibrosis is mediated in male mice and opens the possibility of a molecular pathway mediated by Nkx3.1 that could explain sexual dimorphism in bladder fibrosis. ... Mice were weighed every 2 to 3 days (Fig. 3). As expected, mice in the castration groups had less weight gain compared with the sham groups, as has been reported elsewhere (19, 34, 35). ... Castration Determines the Efficacy of ETAR Blockade in a Mouse Model of Prostate Cancer Bone Metastasis ENDOCRINOLOGY Henry H Moon Katrina L Clines Mark A Cooks Charlotte A Cialek Gregory A Clines Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 (ET-1) is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ETAR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ETAR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that in a mouse model, ETAR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaPM castrate-resistant prostate cancer cell line. The mice were then either treated with the ETAR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared to the zibotentan-treated groups. We report a complex interaction between ETAR and androgen signaling whereby ETAR blockade was most efficacious when combined with complete androgen deprivation. ... Similar effects were observed using rodent models of renal, pulmonary, cardiac, and hepatic fibrosis (Bennett et al., 2014;Fallowfield et al., 2014;Garber et al., 2001;Samuel et al., 2004a;Unemori et al., 1996). Finally, mice deficient in either endogenous relaxin or the relaxin receptor, RXFP1, develop age-related fibrosis in numerous organs (Du et al., 2003; Hewitson et al., 2012; Krajnc-Franken et al., 2004;Samuel et al., 2009;Samuel et al., 2003;Samuel et al., 2005;Samuel et al., 2004b). Therefore, there is considerable in vitro and in vivo evidence that relaxin is an important factor in the progression and treatment of fibrosis. ... Relaxin and extracellular matrix remodeling: Mechanisms and signaling pathways MOL CELL ENDOCRINOL Hooi Hooi Ng Matthew Shen Chrishan S. Samuel Jens Schlossmann Robert G. Bennett Fibrosis is associated with accumulation of excess fibrillar collagen, leading to tissue dysfunction. Numerous processes, including inflammation, myofibroblast activation, and endothelial-to-mesenchymal transition, play a role in the establishment and progression of fibrosis. Relaxin is a peptide hormone with well-known antifibrotic properties that result from its action on numerous cellular targets to reduce fibrosis. Relaxin activates multiple signal transduction pathways as a mechanism to suppress inflammation and myofibroblast activation in fibrosis. In this review, the general mechanisms underlying fibrotic diseases are described, along with the current state of knowledge regarding cellular targets of relaxin. Finally, an overview is presented summarizing the signaling pathways activated by relaxin and other relaxin family peptide receptor agonists to suppress fibrosis. ... Values are denoted as * * p < 0.01 relaxin-null mice provided particularly strong evidence that relaxin functions to protect against fibrosis (Samuel et al. 2007;Bennett 2009). The role of canonical estrogen receptors and estradiol in the development of cardiac, renal, and systemic fibrosis was also evidenced (Pedram et al. 2010; Hewitson et al. 2012; Aida-Yasuoka et al. 2013). Notably, loss of TCs accompanies fibrosis of multiple organs in systemic sclerosis (Manetti et al. 2014). ... Telocytes in the mouse testicular interstitium: implications of G-protein-coupled estrogen receptor (GPER) and estrogen-related receptor (ERR) in the regulation of mouse testicular interstitial cells Article Full-text available PROTOPLASMA Piotr Pawlicki Anna Hejmej Agnieszka Milon Krzysztof Lustofin Barbara Bilinska Telocytes (TCs), a novel type of interstitial cells, are involved in tissue homeostasis maintenance. This study aimed to investigate TC presence in the interstitium of mouse testis. Additionally, inactivation of the G-coupled membrane estrogen receptor (GPER) in the testis was performed to obtain insight into TC function, regulation, and interaction with other interstitial cells. Mice were injected with a GPER antagonist (G-15; 50 μg/kg bw), and the GPER-signaling effect on TC distribution, ultrastructure, and function, as well as the interstitial tissue interaction of GPER with estrogen-related receptors (ERRs), was examined. Microscopic observations of TC morphology were performed with the use of scanning and transmission electron microscopes. Telocyte functional markers (CD34; c-kit; platelet-derived growth factor receptors α and β, PDGFRα and β; vascular endothelial growth factor, VEGF; and vimentin) were analyzed by immunohistochemistry/immunofluorescence and Western blot. mRNA expression of CD34 as well as ERR α, β, and γ was measured by qRT-PCR. Relaxin and Ca2+ concentrations were analyzed by immunoenzymatic and colorimetric assays, respectively. For the first time, we reveal the presence of TCs in the interstitium together with the peritubular area of mouse testis. Telocytes were characterized by specific features such as a small cell body and extremely long prolongations, constituting a three-dimensional network mainly around the interstitial cells. Expression of all TC protein markers was confirmed. Based on scanning electron microscopic observation in GPER-blocked testis, groups of TCs were frequently seen. No changes were found in TC ultrastructure in GPER-blocked testis when compared to the control. However, tendency to TC number change (increase) after the blockage was observed. Concomitantly, no changes in mRNA CD34 expression and increase in ERR expression were detected in GPER-blocked testes. In addition, Ca2+ was unchanged; however, an increase in relaxin concentration was observed. Telocytes are an important component of the mouse testicular interstitium, possibly taking part in maintaining its microenvironment as well as contractile and secretory functions (via themselves or via controlling of other interstitial cells). These cells should be considered a unique and useful target cell type for the prevention and treatment of testicular interstitial tissue disorders based on estrogen-signaling disturbances. ... All animals were castrated before study initiation because castrated male Göttingen Minipigs become more prone to obesity and metabolic changes after castration [31]. Literature state that castration may protect against age-related myocardial fibrosis in mice [46] , but also that castration promotes myocardial fibrosis formation in rodent heart failure models [47,48]. However, to the authors' knowledge, no studies have investigated this relationship in pigs. ... Dietary normalization from a fat, fructose and cholesterol-rich diet to chow limits the amount of myocardial collagen in a Göttingen Minipig model of obesity NUTR METAB Laura Jul Andreasen Simone Krog Trine Pagh Ludvigsen Ole Lerberg Nielsen Lisbeth Høier Olsen Background Dietary interventions have been shown to attenuate some of the myocardial pathological alterations associated with obesity. This study evaluated the effect of dietary normalization from a fat/fructose/cholesterol-rich diet to chow on left ventricular (LV) myocardial fibrosis, fat infiltration and hypertrophy but also the specific influence of obesity, plasma lipids and glucose metabolism markers on heart morphology in a Göttingen Minipig model of obesity. Methods Forty castrated male Göttingen Minipigs were assigned to three groups fed either standard minipig chow (SD, n = 8) for 13 months, fat/fructose/cholesterol-rich diet (FFC, n = 16) for 13 months or fat/fructose/cholesterol-rich diet for 7 months and then changed to standard minipig chow for the remaining 6 months (FFC/SD, n = 16). Body weight, body fat percentage, plasma lipids and glucose metabolism markers were evaluated in all three groups after 6–7 months (prior to diet adjustment for FFC/SD) and again before termination. Further, biochemical quantification of myocardial collagen and triglyceride content, semi-quantitative histological evaluation of fibrosis and fat infiltration and quantitative histological analysis of collagen and cardiomyocyte diameter were performed and heart weight was obtained after termination. Group differences were evaluated using Kruskal-Wallis test and Fisher’s exact test for categorical variables. Pearson correlation analysis was performed to test for correlations between myocardial changes and selected explanatory variables. For non-parametric response variables, a Spearman correlation analysis was applied. Results Myocardial collagen content quantified biochemically was significantly lower in FFC/SD compared to FFC (P = 0.02). Furthermore, dietary normalization from a fat/fructose/cholesterol-rich diet to chow caused stagnation of body weight and body fat percentage, normalized intravenous glucose tolerance index (KG) and plasma lipid levels. Conclusion Dietary normalization led to lower LV collagen content in obese Göttingen Minipigs. Despite gross obesity and significant deteriorations in glucose and lipid metabolism, only mild myocardial changes were found in this model of obesity and therefore further model optimization is warranted in order to induce more severe myocardial changes before dietary or pharmacological interventions. ... 16 In most cases, male mice developed more severe fibrosis than the females, likely due to testosteronerelated effects. 17 Similar effects were seen in mice lacking Rxfp1, which codes for the cognate receptor for relaxin. These Rxfp1 mice developed sex-specific pulmonary fibrosis with age. ... Targeting the Relaxin Pathway for Liver Disease Treatment Robert G Bennett Hepatic fibrosis is a progressive disease with few treatment options outside of transplantation. Relaxin is a member of the insulin/relaxin superfamily of peptide hormones. Originally known for its roles in pregnancy, relaxin promotes reproductive tissue remodelling and regulates vascular changes, including increased arterial compliance and reduced vascular resistance. Outside of pregnancy, relaxin plays a major role in the protection of organs from excess extracellular matrix accumulation, as demonstrated by the relaxin-null mouse, which develops widespread fibrosis with ageing. Relaxin reduces scarring due to excess collagen deposition by inhibiting collagen production while simultaneously promoting its degradation and can reduce established fibrosis in several animal models of extracellular matrix-associated disease, including liver fibrosis. Treatment with relaxin reduces the myofibroblastic phenotype of activated hepatic stellate cells, the major hepatic collagen-producing cell in fibrosis and cirrhosis. Relaxin also has haemodynamic effects, including vasodilation, and can reduce portal hypertension associated with cirrhosis. In this review, a brief overview of hepatic fibrosis and the role of the hepatic stellate cell will be presented, followed by an introduction to relaxin and its actions. The use of relaxin to treat preclinical models of fibrotic diseases, including liver diseases, will also be discussed. Finally, the completed, current, and ongoing clinical trials of relaxin in human disease will be described, followed by the limitations and future directions for the use of relaxin for disease treatment. ... This included a blunting of the TGFβ signaling pathway, known to be important for promoting myofibroblast differentiation (16). Age-related cardiac and renal fibrosis was also attenuated by orchiectomy in male mice (17) . ... Testosterone Modifies Alterations to Detrusor Muscle after Partial Bladder Outlet Obstruction in Juvenile Mice Paula R. Firmiss Diana K. Bowen Natalie Kukulka Edward M. Gong Lower urinary tract symptoms secondary to posterior urethral valves (PUV) arise in boys during adolescence. The reasons for this have previously been attributed to increased urine output as boys experience increased growth. Additionally, there are few choices for clinicians to effectively treat these complications. We formed the new hypothesis that increased androgen levels at this time of childhood development could play a role at the cellular level in obstructed bladders. To test this hypothesis, we investigated the role of testosterone on bladder detrusor muscle following injury from partial bladder outlet obstruction (PO) in mice. A PO model was surgically created in juvenile male mice. A group of mice were castrated by bilateral orchiectomy at time of obstruction (CPO). Testosterone cypionate was administered to a group of castrated, obstructed mice (CPOT). Bladder function was assessed by voiding stain on paper (VSOP). Bladders were analyzed at 7 and 28 days by weight and histology. Detrusor collagen to smooth muscle ratio (Col/SM) was calculated using Masson’s trichrome stain. All obstructed groups had lower max voided volumes (MVV) than sham mice at 1 day. Hormonally intact mice (PO) continued to have lower MVV at 7 and 28 days while CPO mice improved to sham levels at both time points. In accordance, PO mice had higher bladder-to-body weight ratios than CPO and sham mice demonstrating greater bladder hypertrophy. Histologically, Col/SM was lower in sham and CPO mice. When testosterone was restored in CPOT mice, MVV remained low at 7 and 28 days compared to CPO and bladder-to-body weight ratios were also greater than CPO. Histologic changes were also seen in CPOT mice with higher Col/SM than sham and CPO mice. In conclusion, our findings support a role for testosterone in the fibrotic changes that occur after obstruction in male mice. This suggests that while other changes may occur in adolescent boys that cause complication in boys with PUV, the bladder itself responds to testosterone at the cellular level. This opens the door to a new understanding of pathways that influence bladder fibrosis and could lead to novel approaches to treat boys with PUV.	https://www.researchgate.net/figure/Continued_fig2_51746366
BOWYER, GWILYM (1906 - 1965), minister (Congl.) and college principal \| Dictionary of Welsh Biography BOWYER, GWILYM (1906 - 1965), minister (Congl.) and college principal Name: Gwilym Bowyer Date of birth: 1906 Date of death: 1965 Spouse: Prydwen Bowyer (née Harrison) Child: Ann Bowyer Child: Mair Bowyer Child: Gwynn Bowyer Parent: Sarah Bowyer Parent: William Bowyer Gender: Male Occupation: minister (Congl.) and college principal Area of activity: Education; Religion Authors: Robert Tudur Jones, Ioan Wyn Gruffydd Born 7 February 1906 at 74a Chapel St., Ponciau, Rhosllannerchrugog, Denbighshire, son of William Bowyer, miner, and his wife Sarah. He was the fifth of their six children. He received his primary education at the Council School, Ponciau, and then from 1920 to 1928 he worked in a grocer's shop, an experience that he greatly appreciated, but received further education under the guidance of J. Powell Griffiths, minister of the English Baptist church, Grenville Williams, a teacher at the Council School, and especially R.J. Pritchard, his minister at Mynydd Seion Congl. church, Ponciau, where he began to preach in 1923. Gwilym Bowyer entered Bala-Bangor College, where his elder brother Frederick had already been a student for three years and whereJohn Morgan JonesandJ.E. Danielwere lecturers, 27 September 1928 and he graduated B.A. with first-class hons. in philosophy in 1932 and B.D. in 1935 with Church History and Christian Doctrine as his main subjects. He was ordained in Soar, Cwmclydach, Rhondda, 12 September 1935 and was subsequently minister of three very different pastorates, in Cwmclydach, 1935-39, at the Borough, London (1939-43) and Ebeneser, Bangor (1943-46). He married Prydwen Harrison of Penmaen-mawr on 1 October 1935 and they had three children, Gwynn, Mair and Ann. Whilst living in the Rhondda Valley, Bowyer became very aware of the crisis of Wales in the context of the world's anguish, and there were few who could understand or appreciate their minister's strong convictions, especially with regard to the cause of peace and the safeguarding of the Welsh language. He moved to the Borough at the painful and difficult period of war - a period of bombing when women and children were compelled to return to Wales on account of the dangers, as his wife and his son Gwynn were obliged to do. He found in Bangor conditions that were more congenial to his intentions. And when his time as minister came to an end, he continued to serve as a valuable deacon at Ebeneser. He began his work as principal of Bala-Bangor College in October 1946 and remained in post until his death following a heart attack in October 1965. This was the period of his great contribution, not only to his own denomination but to his nation as well. He lectured on Christian Doctrine to his students at Bala-Bangor, and he also was in charge of the student hostel. The wide span of his interests - from women's magazines to the most recent films at the local cinemas - became obvious when he shared the lunch table with his students; he enjoyed reading detective novels. On one particular Sunday, it had been arranged for him to travel with me (Ioan Wyn Gruffudd) to his preaching appointment, but when that became common knowledge amongst the students, there was no end to teasing the Principal what a dangerous driver I was! He possessed a brilliant mind and a quick tongue. He was stronger in analysing and criticising than in constructing. As a theologian he tended towards rather conservative views and the thinkers who influenced him most were Augustine, Luther, Kierkegaard and his professor,J.E. Daniel. He was an interpreter of the Barthian viewpoint, and he valued the individualism of Kierkegaard which explained many aspects that he considered of great importance - his suspicion of every ecclesiastical body, his constant stress on personal responsibility, his unwillingness to be associated with any political party, his fear of the interference of the state. Christianity for Gwilym Bowyer meant an allegiance to Jesus Christ - an unquestionable allegiance. Politically he was a radical, a convinced pacifist and a strong advocate for Welsh-medium education. He made a substantial contribution towards making it possible for theological students in the University of Wales to follow courses and take their examinations in Welsh. Gwilym Bowyer died in Bangor on 5 October 1965 aged 59. He was buried 8 October at New Cemetery, Bangor. Authors Reverend Principal Robert Tudur Jones , (1921 - 1998) Ioan Wyn Gruffydd Sources W. Eifion Powell, Bywyd a gwaith Gwilym Bowyer (Swansea 1968) Portreadau'r Faner , ii, 36-8 Y Dysgedydd , November/December 1965 Y Tyst , 4 November 1965 Additional Links	https://biography.wales/article/s2-BOWY-GWI-1906?lang%5B%5D=en&order=asc&page=8&query=John%20Gibson&rows=12&sort=sort_name
Molecules \| Free Full-Text \| Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII A series of new 3- and 7-substituted sulfocoumarins was obtained by several cyclization reactions and subsequent derivatization for screening as prodrug inhibitors of the human (h) cancer-associated carbonic anhydrases (CAs) IX and XII. All products were ineffective inhibitors against the off-target hCA I and II, whilst hCAs IX and XII were inhibited with inhibition constants (KIs) spanning from low nanomolar to the high micromolar range, according to the sulfocoumarin derivatization pattern. In particular, sulfocoumarin 15 turned out to be the most potent and selective inhibitor herein reported (hCA I and II: KI > 100 µM; hCA IX: KI = 22.9 nM; hCA XII: KI = 19.2 nM). Considering that hCA IX and XII validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here may be useful for identifying suitable drug candidates for clinical trials. Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII by Simone Giovannuzzi 1 , Clemente Capasso 2 , Alessio Nocentini 1,* and Claudiu T. Supuran 1,* Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy Authors to whom correspondence should be addressed. Molecules 2022 , 27 (13), 4076; https://doi.org/10.3390/molecules27134076 Received: 30 May 2022 / Revised: 15 June 2022 / Accepted: 23 June 2022 / Published: 24 June 2022 (This article belongs to the Special Issue Enzyme Inhibitors: Design, Synthesis and Biological Evaluation ) Abstract : A series of new 3- and 7-substituted sulfocoumarins was obtained by several cyclization reactions and subsequent derivatization for screening as prodrug inhibitors of the human (h) cancer-associated carbonic anhydrases (CAs) IX and XII. All products were ineffective inhibitors against the off-target hCA I and II, whilst hCAs IX and XII were inhibited with inhibition constants (K I s) spanning from low nanomolar to the high micromolar range, according to the sulfocoumarin derivatization pattern. In particular, sulfocoumarin 15 turned out to be the most potent and selective inhibitor herein reported (hCA I and II: K I > 100 µM; hCA IX: K I = 22.9 nM; hCA XII: K I = 19.2 nM). Considering that hCA IX and XII validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here may be useful for identifying suitable drug candidates for clinical trials. Keywords: carbonic anhydrase ; cancer ; sulfocoumarin 1. Introduction Carbonic anhydrases are a ubiquitous metalloenzyme family present in all living organisms. Until now, eight genetically distinct CA subfamilies have been identified [ 1 , 2 , 3 ]. In mammals, 16 CA isoforms belonging to α-CAs are characterized, and in humans 15 CA (hCA) isoforms are expressed, showing differences in kinetic properties, subcellular localization and tissue distributions [ 4 , 5 ]. Twelve such isoforms are catalytically active (CAs I–IV, VA, VB, VI, VII, IX, XII and XIV), whereas the remaining three isoforms (VIII, X, XI), called CA-related proteins (CARPs), have no activity. hCAs can be further categorized into four different subsets depending on their subcellular localization as cytosolic (hCA I, II, III, VII, VIII, X, XI, XIII), mitochondrial (hCA VA and VB), secreted (hCA VI) and membrane-bound (hCA IV, IX, XII, and XIV) [ 5 , 6 , 7 ]. Four distinct CA inhibition mechanisms have been reported and detailed to date with both kinetic and X-crystallographic studies [ 8 , 9 ]. They include (a) the metal ion binders (anions, sulfonamides and their bioisosteres, dithiocarbamates, xanthates, etc.); (b) compounds that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines); (c) compounds occluding the active site entrance, such as coumarins and their isosteres (sulfocoumarins); and (d) compounds binding out of the active site, such as an aromatic carboxylic acid derivative [ 8 , 9 , 10 ]. Coumarins, such as 1 (natural product isolated from the Australian plant Leionema ellipticum) and 2 (the simple unsubstituted coumarin), were discovered as a new chemotype that can inhibit the metalloenzyme carbonic anhydrase a decade ago [ 11 , 12 , 13 ]. Many differently substituted coumarins were subsequently screened for their inhibitory activity against all the 13 catalytically active mammalian CA isoforms, CA I-VII, IX, XII-XV [ 14 , 15 , 16 , 17 , 18 ]. Many of these isoforms are established drug targets for designing agents with various applications, such as diuretics, antiglaucoma drugs, anticonvulsants, antiobesity agents or antitumor drugs/cancer diagnostic tools [ 17 , 18 ]. To explain the inhibitor mechanism of coumarins 1 and 2 , they were cocrystallized with human CA II, and the electron density data showed the presence of the hydrolyzed derivatives 4 and 5 , respectively ( Figure 1 ). The most notable aspect of this inhibition mechanism is the fact that the 4 and 5 occlude the enzyme active site binding at the entrance of the cavity. Over the last several years, a rather large number of new classes of CAIs were reported, starting from coumarins as the lead, among which are thiocoumarins, sulfocoumarins, 2-thioxo-coumarins, coumarin oximes, 5-/6-membered/thio)lactones, etc. [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]; coumarins and sulfocoumarins were also shown to be isoform-selective CA inhibitors. Particularly, in 2012, the sulfocoumarins were identified as CAIs using kinetic and X-ray crystallographic studies, in which it has been also revealed that although structurally related to the coumarins, sulfocoumarins possess a different CA inhibition mechanism ( Figure 2 ) [ 29 ]. As reported in Figure 1 for compound 3 , sulfocoumarin were hydrolyzed by α-CAs to give trans-2-hydroxyphenyl-ω-ethenylsulfonic acid. Afterwards, the formed sulfonic acid binds to the CA II active site by anchoring the SO 3 H group to the zinc-coordinated water molecule/hydroxide ion ( Figure 2 ) [ 30 ]. After this discovery, many more derivatives were synthesized and analyzed for their interaction with different CA isoforms [ 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. 2. Results 2.1. Chemistry Due to the difficulties in designing and synthesizing selective sulfonamide inhibitors against each isoform, such as SLC-0111 ( Figure 3 ) [ 38 ], a potent and selective zinc binder CAI against hCA IX and XII, scientists opted for the development of novel chemotypes among which are sulfocoumarins, the preferred CAI scaffold adopted in this project. The general strategy of Zalubovskis group [ 16 , 29 ], for the preparation of 6-substituted sulfocoumarins and validated by Nocentini et al. [ 36 ], in 2015, for the synthesis of 7-substituted such derivatives, was applied in this manuscript to extend the structure-activity relationships, whereas the general strategy of Liu’s group [ 39 ], for the designed of 3-substituted sulfocoumarins, was validated in this project in order to synthesize 3-amido derivatives for the first time. To start, 2-hydroxy-4-methoxybenzaldehyde 7 or 2′-hydroxy-4′-methoxyacetophenon 8 were synthesized with the sulfocoumarin scaffold 9 and 10 . After that, their phenol moiety was released in the presence of BBr 3 in dry DCM to obtain 11 and 12 in high yield and high purity. Finally, a nucleophilic substitution was performed in the presence of different benzyl bromides with K 2 CO 3 as a base in dry DMF at RT to give compounds 13 – 22 ( Scheme 1 ). Compound 23 (2-bromobenzenesulfonyl chloride) was reacted with 3-methoxyphenol in dry DCM in the presence of Et 3 N as a base to obtain intermediate 24 ; after that, a cyclization was performed in dry DMA with Pd(OAc) 2 as a catalyst at 150 °C to give the sulfocoumarin 25 . To prepare the next reaction, the methoxy group was released in the presence of BBr3 in dry DCM between − 10 °C and RT to synthesize intermediate 26, and then a nucleophilic substitution was performed in dry DMF at RT with K 2 CO 3 as a base to give us compounds 27 – 31 ( Scheme 2 ). A cyclization reaction was performed between starting materials 32 , or salicylaldehyde, or 33 , 4-methoxysalicylaldehyde, and ethyl 2-chlorosulfonylacetate in DCE at 90 °C in the presence of dry pyridine as a base; after that, the obtained ethyl esters ( 34 , 35 ) were hydrolyzed in EtOH and NaOH 5M at reflux to release the carboxylic acid moiety of compounds 36 and 37 . Finally, a coupling reaction was performed in dry DMF between appropriate anilines and intermediates 36 and 37 to give us products 38 – 47 with an amide as linker ( Scheme 3 ). 2.2. Carbonic Anhydrase Inhibition Sulfocoumarins 13 – 22 , 27 – 31 , 38 – 47 were screened in vitro for the inhibition of four physiologically relevant hCA isoforms, the cytosolic hCAI and II and the trans-membrane tumor-associated hCA IX and XII [ 4 , 5 , 6 , 8 , 40 , 41 , 42 , 43 ]; acetazolamide (AAZ) was used as standard CAI. CA I is the main off-target isoform for most therapeutic applications of CAIs, whilst CA II is considered off-target in many pathologies to reduce side effects resulting from systematic CA inhibition as much as possible. Table 1 shows the inhibition data obtained after a period of incubation of 6 h of the enzyme and inhibitors. Noteworthily, the assay inhibition performed within the usual 15 min incubation period (as for the sulfonamides) led to the very weak inhibition constants (data not shown) [ 43 ]. For this reason, we herein report a 6 h incubation time instead. The following structure-activity relationship (SAR) can be gathered from the inhibition data reported in Table 1 . The following structure-activity relationships (SAR) should be noted: - According to the previous reports [ 31 , 32 , 33 , 35 ], isoform hCA I was not inhibited by a large number of substituted sulfocoumarins; however, on the other side, the simplest sulfocoumarin as 11 , 12 , 26 , 36 and 37 showed a weak inhibitory potency. It is likely that compounds 36 (K I = 4625 nM) and 37 (K I = 7139 nM) have a low micromolar inhibition due to the presence of the carboxylic acid moiety, which may work by binding to the H 2 O molecule coordinated with Zn 2+ in the active site [ 45 , 46 ]. - Similar to hCA I, for hCA II we did not observe any inhibition by all the substituted sulfocoumarines, except for 9 – 12 , 26 , 36 and 37 . Compounds 11 and 12 showed a low micromolar inhibition, respectively K I = 2896 nM and K I = 3857 nM, whilst analogs 9 and 10 have the K I in the medium micromolar range, which can likely be associated to the presence of a free phenol moiety on products 11 and 12 , which is protected in 9 and 10 . - The target hCA IX resulted in being the most inhibited isoforms by sulfocoumarins reported here. Considering the three different types of products, type 1 shows the most efficient inhibition against this isozyme, with K I values in the low-medium nanomolar range, between 14.3 nM and 97.2 nM. In detail, the first-in-class compound in term of inhibition potency is the number 19 , which shows a fluorine atom in para position on the benzyloxy moiety and a methyl group as R1 substituent (K I = 14.3 nM), whilst the elimination of the benzyloxy moiety ( 10 ) gives us the less effective compound (K I = 97.2 nM). Interestingly, the general behaviour of these compounds owned on type 1 was that, indeed, the insertion of the methyl group as an R1 substituent produced a worsening of the K I values; in fact, it was demonstrated by products 16 (K I = 68.9 nM) and 17 (K I = 40.2 nM) compared to the corresponding 21 (K I = 89.7 nM) and 22 (K I = 61.4 nM). This rule seems to be broken only by derivatives 18 (K I = 49.8 nM) and 19 (K I = 14.3 nM), which resulted in better inhibitors with respect to analogs 13 (K I = 55.6 nM) and 14 (K I = 22.9 nM). Sulfocoumarines belonging to type 2, even with a benzyloxy moiety, suffered from the addition of a further aromatic ring on the scaffold that improves the steric hindrance on the sulfonate ring, which induced a worsening of the K I values. Indeed, the inhibition constants are in the high nanomolar range, between 615.1 nM and 1286 nM. The best compound result to be 29 (K I = 615.1 nM) with a fluorine atom in meta position on the benzyloxy moiety. The movement of the R1 group from meta to para position produces a worsening of the K I values. Examples are 29 (K I = 615.1 nM) and 31 (K I = 898.1 nM), respectively, with 28 (K I = 754.9 nM) and 30 (K I = 964.7 nM). Interestingly, it is a significant decline of the inhibition data of the sulfocoumarines belonging to type 3, with an amide as linker on position 3 of the sulfonate ring. It is likely that the presence of bulky groups with the CAI portion results in a worsening of the K I values, in a micromolar range between 499.7 nM and 64380 nM. The most potent compounds of this class are 43 (K I = 2429 nM) and the simplest sulfocoumarines 34 (K I = 1631 nM), 35 (K I = 1085 nM), 36 (K I = 499.7 nM) and 37 (K I = 853.7 nM) showed an ester or carboxylic acid moieties in position 3. Sulfocoumarines with a bromine atom in a meta position as the R2 group ( 40 , 45 ) resulted in the worst values of inhibition, respectively K I = 40,840 nM and 64,380 nM. The hCA XII resulted in becoming the second most efficiently inhibited isoform by sulfocoumarines, with it being particularly possible to observe a similar trend of the K I values as already shown for the hCA IX. Considering products belonging to type 1, they show the most potent inhibition for this isoform, between 8.6 nM and 136.0 nM. The presence of a methyl group in R1 position produced an improvement of the K I values for derivatives 18 (K I = 8.6 nM), without substituent on the benzyloxy moiety, and 20 (K I = 25.7 nM), with fluorine in the meta position. For all the other products, a worsening of Ki values, such as for 14 (K I = 19.2 nM) and 19 (K I = 41.7 nM), or for 11 (K I = 42.5 nM) and 12 (K I = 55.0 nM), was observed. On the other side, the movement of the fluorine atom from meta to para position ( 19 ) resulted in a K I value of 41.7 nM. Regarding the nitro group, compound 17 (K I = 77.5 nM) with R2 group in a meta position show an improvement in term of inhibition than its analogue 16 (K I = 94.7 nM) with R2 group in a para position. For sulfocoumarines 18 – 22 , the fluorine atom resulted in being better than nitro group as a substituent on the benzyloxy moiety; indeed, 19 (K I = 41.7 nM) and 20 (K I = 25.7 nM) are strongest inhibitors in term of potency than 21 (K I = 100.8 nM) and 22 (K I = 136.0 nM). For compounds belonging to type 2, the addition of an aromatic ring on the sulfocoumarin scaffold determines a worsening of the K I values in the medium–high nanomolar range, between 414.2 nM and 1369 nM. The presence of a bulky group such as the nitro group leads to a worsening in terms of inhibition potency, particularly products 30 and 31 have K I of 1056 nM and 1369 nM. The simplest sulfocoumarin with a free phenol moiety, 26 , showed the best inhibition for type 2 series, 414.2 nM. Sulfocoumarins belonging to type 3 that present a further enhancement of the steric hindrance on the sulfonate ring, have K I values in the range of micromolar (K I = 408.2–78,320 nM). Compound 40 and 45 , with a bromine atom in the meta position, show the worst inhibition potencies on hCA XII, respectively 78,320 nM and 76,540 nM; on the other side, sulfocoumarines with the less bulky group in R 2 position result in being a good inhibitor on this isoform, while, indeed, 38 and 43 with a hydrogen atom show K I values in the range of low micromolar—respectively, 2301 nM and 1594 nM. The best inhibitors belonging to the type 3 series are compounds 36 (K I = 563.7 nM) and 37 (K I = 408.2 nM), likely thanks to the free carbocylic acid moiety. 3. Materials and Methods 3.1. Chemistry Anhydrous solvents and all reagents were purchased from Merck, Fluorochem and TCI. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance (1H-NMR, 13C-NMR,) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d 6 . Chemical shifts are reported in parts per million (ppm), and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet; dd, doublet of doublets. The assignment of exchangeable protons was confirmed by the addition of D 2 O. Analytical thin-layer chromatography (TLC) was carried out on Sigma Aldrich silica gel F-254 plates. Flash chromatography purifications were performed on Sigma Aldrich Silica gel 60 (230–400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane or MeOH/DCM were used as eluents. Melting points (mp) were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 appa-ratus and are uncorrected. The solvents used in mass spectrometry analysis were acetone, acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich (Milan, Italy), and mQ water 18 MΩ cm, obtained from Millipore’s Simplicity system (Milan, Italy). The HPLC-MS and MS/MS analysis was carried out using a Varian 500-MS ion trap system (Palo Alto, CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Electrospray source (ESI) operating in negative ions. Stock solutions of analytes were prepared in acetone at 1.0 mg mL-1 and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of mQ water:acetonitrile 1:1 ( v / v ) up to a concentration of 1.0 µg mL −1 . The mass spectra of each analyte were acquired by introducing, via syringe pump at 10 µL min −1 , the working solution. Raw data were collected and processed by Varian Workstation Vers. 6.8 software. General synthetic procedure for 7-methoxybenzo[e][1,2]oxathiine 2,2-dioxide (9) and 7-methoxy-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide (10) Et 3 N (1.5 equiv.) and Mesyl chloride (1.5 equiv.) were added slowly to a solution of 7 or 8 (3 g, 1 equiv.) in dry DCM (20 mL) at 0 °C under nitrogen atmosphere. The solution was stirred for 2 h at RT. Slush was added to the mixture and the reaction mixture was extracted in DCM (3 × 25 mL), dried with Na 2 SO 4 , filtered and evaporated to give us a pale yellow oil. DBU (1.2 equiv.) was added dropwise to a solution of the oil in dry DCM (20 mL) at 0 °C under nitrogen atmosphere. The solution was stirred o.n. at RT. Slush was added slowly and the reaction mixture was extracted in DCM (3 × 25 mL). The collected organic phases were dried with Na 2 SO 4 , filtered and evaporated under vacuum to give a brown oil. POCl 3 (1.5 equiv.) was added dropwise to a solution of the oil in dry Py (3 mL) at 0 °C under nitrogen atmosphere. The solution was stirred on at RT. Ice was added slowly to the reaction mixture and the resulting precipitate was filtered to give intermediates 9 and 10 as a light brown powder in high yield. 9 and 10 were purified by silica gel chromathography column (EtOAc/Hexane: from 20% to 60% v / v ) to give white-grey powder. 7-Methoxybenzo[e][1,2]oxathiine 2,2-dioxide ( 9 ) Compound 9 was obtained according to the general procedure reported earlier with 4-methoxysalicyl aldehyde ( 7 ) as starting material. Yield 81%; m.p. 111–112 °C; silica gel TLC Rf 0.82 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.65 (d, J = 10.0 Hz, 1H, Ar- H ), 7.63 (d, J = 8.4 Hz, 1H, Ar- H ), 7.32 (d, J = 10.0 Hz, 1H, Ar- H ), 7.09 (d, J = 2.2 Hz, 1H, Ar- H ), 7.01 (dd, J = 8.4 2.2 Hz, 1H, Ar- H ), 3.88 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 163.3, 153.3, 137.4, 132.0, 120.0, 113.8 112.8, 104.7, 57.0. 7-Methoxy-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 10 ) Compound 10 was obtained according to the general procedure reported earlier with 2-hydroxy-4-methoxyacetophenon ( 8 ) as starting material. Yield 76%; m.p. 151–153 °C; silica gel TLC Rf 0.79 (MeOH/DCM 5% v / v ): 7.73 (d, J = 8.2 Hz, 1H, Ar- H ), 7.24 (s, 1H, Ar- H ), 7.10 (s, 1H, Ar- H ), 7.05 (d, J = 8.2 Hz, 1H, Ar- H ), 3.90 (s, 3H, CH 3 ), 2.37 (s, 3H, C H 3 ); δ H (400 MHz, DMSO-d 6 ); δ C (400 MHz, DMSO-d 6 ): 157.5, 154.3, 142.7, 126.8, 119.3, 107.6, 107.3, 106.8, 558, 19.9. Synthetic procedure for 3-methoxyphenyl 2-bromobenzenesulfonate (24) Et 3 N (2 equiv.) and 2-bromobenzenesulfonyl chloride ( 23 ) (1 equiv.) were added slowly to a solution of 3-methoxyphenol (0.5 g, 1.1 equiv.) in dry DCM at 0 °C under nitrogen atmosphere. The solution was stirred for 4 h at RT. Slush and HCl 2M were added to pH = 4 and the reaction mixture was extracted in DCM (3 × 15 mL). The collected organic phases were washed with K 2 CO 3 s.s. (2 × 10 mL), dried with Na 2 SO 4 , filtered and evaporated under vacuum to give products 24 as white powder in high yield and purity. Yield 96%; m.p. 163–166 °C; silica gel TLC Rf 0.45 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.08 (d, J = 8.4 Hz, 1H, Ar- H ), 7.99 (d, J = 8.4 Hz, 1H, Ar- H ), 7.75 (dd, J = 7.4 Hz, 1H, Ar- H ), 7.65 (dd, J = 7.4 Hz, 1H, Ar- H ), 7.33 (dd, J = 7.1 Hz, 1H, Ar- H ), 6.93 (d, J = 7.1 Hz, 1H, Ar- H ), 6.70 (m, 2H, Ar- H ), 3.73 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 162.0, 152.7, 146.8, 134.7, 131.7, 134.7, 131.1, 130.3, 129.0, 115.6, 106.9, 102.0, 55.8. Synthetic procedure for 3-methoxydibenzo[c,e][1,2]oxathiine 6,6-dioxide (25) Potassium acetate (2 equiv.) and Pd(OAc) 2 (0.01 equiv.) were added to a solution of 24 (1.2 g, 1 equiv.) in dry DMA (5 mL) under nitrogen atmosphere. The solution was heated at 150 °C for 4 h. The Resulting mixture was cooled in ice bath and slush was added. The suspension was extracted in EtOAc (3 × 20 mL), and the collected organic phases were washed with brine (3 × 50 mL), dried with Na 2 SO 4 , filtered and evaporated under vacuum to give 25 Yield 93%; m.p. 180–183 °C; silica gel TLC Rf 0.45 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.25 (dd, J = 8.3 Hz, 2H, Ar-H), 8.07 (dd, J = 8.3 Hz, 2H, Ar-H), 7.95 (m, 1H, Ar-H), 7.72 (m, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.15 (dd, J = 8.3 Hz, 1H, Ar-H), 3.92 (s, 3H, CH 3 ); δ C (400 MHz, DMSO-d 6 ): 160.9, 157.2, 142.4, 134.4, 133.0, 130.3, 128.9, 128.6, 122.7, 119.4, 102.5, 55.8. General syndhetic procedure for 7-hydroxybenzo[e][1,2]oxathiine 2,2-dioxide (11),7-hydroxy-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide (12) and 3-hydroxydibenzo[c,e][1,2]oxathiine 6,6-dioxide (26) BBr 3 (5 equiv.) was added dropwise and slowly to a solution of 9 , 10 or 25 (2 g, 1 equiv.) in dry DCM (15 mL) at −20 °C under nitrogen atmosphere. The solution was stirred 24 h at RT. Ice was added slowly to the solution and the precipitate was filtered. The resulting powder was dissolved in K 2 CO 3 s.s. (15 mL) and was filtered off. The H 2 O-phase was acidified with HCl 6 M to pH = 4 and the product was extracted in EtOAc (3 × 20 mL), dried with Na 2 SO 4 , filtered and evaporated under vacuum to give products 11 , 12 and 26 as white powder in good yield and high purity. 7-Hydroxybenzo[e][1,2]oxathiine 2,2-dioxide ( 11 ) Compound 11 was obtained according to the general procedure reported earlier with 9 as starting material. Yield 72%; m.p. 157–159 °C; silica gel TLC Rf 0.41 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.81 (s, 1H, exchange with D 2 O, O H ), 7.59 (d, J = 9.8 Hz, 1H, Ar- H ), 7.52 (d, J = 8.9 Hz, 1H, Ar- H ), 7.22 (d, J = 9.8 Hz, 1H, Ar- H ), 6.82 (dd, J = 8.9 1.4 Hz, 1H, Ar- H ), 6.75 (d, J = 1.4 Hz, 1H, Ar- H ); δ C (400 MHz, DMSO-d 6 ): 159.8, 155.5, 133.4, 131.2, 126.7, 109.1, 108.4, 106.5. 7-Hydroxy-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 12 ) Compound 12 was obtained according to the general procedure reported earlier with 10 as starting material. Yield 70%; m.p. 172–175 °C; silica gel TLC Rf 0.38 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.84 (s, 1H, exchange with D 2 O, O H ), 7.63 (d, J = 8.7 Hz, 1H, Ar- H ), 7.14 (s, 1H, Ar- H ), 6.87 (d, J = 8.7 Hz, 1H, Ar- H ), 6.77 (s, 1H, Ar- H ), 2.34 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 159.0, 155.1, 142.7, 127.2, 119.3, 108.4, 107.9, 106.2, 19.9. 3-Hydroxydibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 26 ) Compound 26 was obtained according to the general procedure reported earlier with 25 as starting material. Yield 86%; m.p. 181–182 °C; silica gel TLC Rf 0.44 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.68 (s, 1H, exchange with D 2 O, O H ), 8.19 (d, J = 7.9 Hz, 1H, Ar- H ), 8.12 (d, J = 7.9 Hz, 1H, Ar- H ), 8.0 (d, J = 7.4 Hz, 1H, Ar- H ), 7.92 (dd, J = 7.4 Hz, 1H, Ar- H ), 7.68 (dd, J = 7.4 Hz, 1H, Ar- H ), 6.97 (d, J = 7.4 Hz, 1H, Ar- H ), 6.88 (s, 1H, Ar- H ); δ C (400 MHz, DMSO-d 6 ): 158.8, 157.6, 142.4, 134.4, 134.4, 133.0, 130.7, 128.6, 122.3, 119.8, 109.0, 104.1. General synthetic procedure for products 13–22, 27–31 K 2 CO 3 (1.05 equiv.) and the appropriate benzyl bromide (0.95 equiv.) were added to a solution of 11 , 12 or 26 (0.15 g, 1 equiv.) in dry DMF (2 mL) under nitrogen atmosphere. The suspension was stirred o.n. at RT. The reaction mixture was quenched with slush and the H 2 O-phase was extracted in EtOAc (3 × 25 mL). The organic phases were collected and were washed with NaOH 5M (3 × 10 mL) and brine (3 × 50 mL), then organic phase was dried with Na 2 SO 4 , filtered and evaporated under vacuum to obtain products as white powder in high yield and purity. 7-(Benzyloxy)benzo[e][1,2]oxathiine 2,2-dioxide (13) Compound 13 was obtained according to the general procedure reported earlier with benzyl bromide as starting material. Yield 74%; m.p. 165–167 °C; silica gel TLC Rf 0.45 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.65 (d, J = 9.1 Hz, 2H, Ar- H ), 7.41 (m, 6H, Ar- H ), 7.18 (s, 1H, Ar- H ), 7.08 (d, J = 8.5 Hz, 1H, Ar- H ), 5.23 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 162.4, 153.3, 137.5, 137.1, 132.2, 129.6, 129.2, 129.0, 120.3, 114.5, 113.1, 105.5, 71.1; m / z (ESI negative): 286.9 [M − H] − , m / z (ESI positive): 289.0 [M + H] + . 7-((4-Fluorobenzyl)oxy)benzo[e][1,2]oxathiine 2,2-dioxide (14) Compound 14 was obtained according to the general procedure reported earlier with 4-fluorobenzyl bromide as starting material. Yield 77%; m.p. 197–199 °C; silica gel TLC Rf 0.39 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.68 (s, 1H, Ar- H ), 7.66 (d, J = 2.5 Hz, 1H, Ar- H ), 7.58 (m, 2H, Ar- H ), 7.34 (d, J = 10.3 Hz, 1H Ar- H ), 7.28 (dd, J = 8.9 Hz, 2H, Ar- H ), 7.20 (d, J = 2.3 Hz, 1H, Ar- H ),7.10 (dd, J = 8.9 2.3 Hz, 1H, Ar- H ), 5.24 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 164.2 (d, J 1 = 244.0 Hz), 162.3, 153.3, 137.5, 133.4, 132.2, 131.4 (d, J 3 = 8.4 Hz), 120.3, 116.5 (d, J 2 = 21.5 Hz), 114.5, 113.1, 105.7, 70.4; m / z (ESI negative): 304.9 [M − H] − , m / z (ESI positive): 307.0 [M + H] + . 7-((3-Fluorobenzyl)oxy)benzo[e][1,2]oxathiine 2,2-dioxide ( 15 ) Compound 15 was obtained according to the general procedure reported earlier with 3-fluorobenzyl bromide as starting material. Yield 76%; m.p. 180–183 °C; silica gel TLC Rf 0.38 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.68 (m, 2H, Ar-H), 7.51 (m, 2H, Ar-H), 7.36 (m, 3H, Ar-H), 7.21 (s, 1H, Ar-H), 7.12 (d, J = 7.1 Hz, 1H, Ar-H), 5.29 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 164.4 (d, J 1 = 241.1 Hz), 62.1, 153.3, 140.0 (d, J 3 = 7.3 Hz), 137.5, 132.2, 131.6 (d, J 3 = 7.6 Hz), 124.8 (d, J 4 = 2.4 Hz), 120.4, 116.0 (d, J 2 = 20.4 Hz), 115.6 (d, J 2 = 21.7 Hz), 114.5, 113.3, 105.7, 70.2; m / z (ESI negative): 304.9 [M − H] − , m / z (ESI positive): 307.0 [M + H] + . 7-((4-Nitrobenzyl)oxy)benzo[e][1,2]oxathiine 2,2-dioxide ( 16 ) Compound 16 was obtained according to the general procedure reported earlier with 4-nitrobenzyl bromide as starting material. Yield 82%; m.p. 215–217 °C; silica gel TLC Rf 0.48 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.32 (d, J = 8.6 Hz, 2H, Ar- H ), 7.78 (d, J = 8.6 Hz, 2H, Ar- H ), 7.70 (d, J = 8.4 Hz, 1H, Ar- H ), 7.68 (d, J = 10.0 Hz, 1H, Ar- H ), 7.36 (d, J = 10.0 Hz, 1H, Ar- H ), 7.23 (d, J = 2.3 Hz, 1H, Ar- H ), 7.14 (dd, J = 8.4 2.3 Hz,1H, Ar- H ), 5.29 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 161.9, 153.3, 148.2, 144.9, 137.5, 132.3, 129.5, 124.7, 120.5, 114.5, 113.4, 105.8, 69.8; m / z (ESI negative): 331.9 [M − H] − , m / z (ESI positive): 334.0 [M + H] + . 7-((3-nitrobenzyl)oxy)benzo[e][1,2]oxathiine 2,2-dioxide ( 17 ) Compound 17 was obtained according to the general procedure reported earlier with 3-nitrobenzyl bromide as starting material. Yield 81%; m.p. 200–202 °C; silica gel TLC Rf 0.51 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.37 (s, 1H, Ar-H) 8.2 (d, J = 8.1 Hz, 1H, Ar-H), 7.96 (d, J = 8.1 Hz, 1H, Ar-H), 7.71 (m, 3H, Ar-H), 7.34 (d, J = 10.3 Hz, 1H, Ar-H), 7.22 (d, J = 2.8 Hz, 1H, Ar-H), 7.12 (dd, J = 8.3 2.8 Hz, 1H, Ar-H), 5.40 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 162.0, 153.3, 148.9, 139.5, 137.5, 135.4, 132.3, 131.2, 124.1, 123.4, 120.5, 114.5, 113.4, 105.8, 69.8; m / z (ESI negative): 332.0 [M − H] − , m / z (ESI positive): 334.0 [M + H] + . 7-(benzyloxy)-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 18 ) Compound 18 was obtained according to the general procedure reported earlier with benzyl bromide as starting material. Yield 83%; m.p. 174–175 °C; silica gel TLC Rf 0.41 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.73 (d, J = 9.0 Hz, 1H, Ar- H ), 7.51 (d, J = 7.1 Hz, 2H, Ar- H ), 7.45 (dd, J = 7.1 Hz, 2H, Ar- H ), 7.40 (q, J = 7.1 Hz, 1H, Ar- H ), 7.23 (s, 1H, Ar- H ), 7.19 (d, J = 2.1 Hz, 1H, Ar- H ), 7.12 (dd, J = 9.0 2.1 Hz, 1H, Ar- H ), 5.27 (s, 2H, C H 2 ), 2.37 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 162.3, 152.4, 146.6, 137.1, 129.6, 129.3, 129.2, 129.0, 117.3, 114.7, 114.3, 105.7, 71.1, 20.0; m / z (ESI negative): 300.9 [M − H] − , m / z (ESI positive): 303.0 [M + H] + . 7-((4-fluorobenzyl)oxy)-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 19 ) Compound 19 was obtained according to the general procedure reported earlier with 4-fluorobenzyl bromide as starting material. Yield 73%; m.p. 195–198 °C; silica gel TLC Rf 0.43 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 7.74 (d, J = 8.9 Hz, 1H, Ar- H ) 7.57 (m, 2H, Ar- H ), 7.28 (m, 3h, Ar- H ), 7.24 (s, 1H, Ar- H ), 7.19 (d, J = 2.1 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.8 2.1 Hz, 1H, Ar- H ), 5.25 (s, 2H, C H 2 ), 2.37 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 164.27 (d, J 1 = 214.7 Hz), 162.2, 152.4, 164.5, 133.4 (d, J 4 = 2.5 Hz), 131.3 (d, J 2 = 8.8 Hz), 129.3, 117.4, 116.5 (d, J 2 = 20.7 Hz), 114.7, 114.3, 105.7, 70.3, 20.0; m / z (ESI negative): 318.9 [M − H] − , m / z (ESI positive): 321.0 [M + H] + . 7-((3-fluorobenzyl)oxy)-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 20 ) Compound 20 was obtained according to the general procedure reported earlier with 3-fluorobenzyl bromide as starting material. Yield 75%; m.p. 214–215 °C; silica gel TLC Rf 0.55 (EtOAc/Hexane 50% v / v ): 7.74 (d, J = 9.0 Hz, 1H, Ar- H ), 7.50 (m, 1H, Ar- H ), 7.35 (m, 2H, Ar- H ), 7.25 (m, 2H, Ar- H ), 7.20 (d, J = 2.2 Hz, 1H, Ar- H ), 7.13 (dd, J = 8.3 2.2 Hz, 1H, Ar- H ), 5.30 (s, 2H, C H 2 ), 2.37 (s, 3H, C H 3 ); δ H (400 MHz, DMSO-d 6 ); δ C (400 MHz, DMSO-d 6 ): 164.5 (d, J 1 = 168.2 Hz), 162.0, 152.4, 146.5, 140.1 (d, J 3 = 7.9 Hz), 131.6 (d, J 3 = 9.6 Hz), 129.4, 124.8 (d, J 4 = 2.5 Hz), 117.4, 116.0 (d, J 2 = 19.6 Hz), 115.6 (d, J 2 = 20.5), 114.8, 114.3, 105.8, 70.2, 20.0; m / z (ESI negative): 318.9 [M − H] − , m / z (ESI positive): 321.0 [M + H] + . 7-((4-nitrobenzyl)oxy)-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 21 ) Compound 21 was obtained according to the general procedure reported earlier with 4-nitrobenzyl bromide as starting material. Yield 64%; m.p. 247–249 °C; silica gel TLC Rf 0.44 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.27 (d, J = 8.9 Hz, 2H, Ar- H ), 7.37 (d, J = 8.9 Hz, 2H, Ar- H ), 7.70 (d, J = 8.4 Hz, 1H, Ar- H ), 7.20 (s, 1H, Ar- H ), 7.14 (d, J = 2.2 Hz, 1H, Ar- H ), 7.10 (dd, J = 8.4 2.2 Hz,1H, Ar- H ), 5.41 (s, 2H, C H 2 ), 2.32 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 161.8, 152.4, 148.2, 146.5, 145.0, 132.8, 129.5, 124.7, 117.5, 115.0, 114.3, 105.8, 69.8, 20.0; m / z (ESI negative): 345.9 [M − H] − , m / z (ESI positive): 348.0 [M + H] + . 7-((3-nitrobenzyl)oxy)-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide ( 22 ) Compound 22 was obtained according to the general procedure reported earlier with 3-nitrobenzyl bromide as starting material. Yield 89%; m.p. 239–241 °C; silica gel TLC Rf 0.57 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.34 (s, 1H, Ar- H ), 8.21 (d, J = 8.1 Hz, 1H, Ar- H ), 7.93 (d, J = 8.1 Hz,1H, Ar- H ), 7.73 (dd, J = 8.1 Hz, 1H, Ar- H ), 7.71 (d, J = 8.3 Hz, 1H, Ar- H ), 7.20 (s, 1H, Ar- H ), 7.19 (d, J = 2.1 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.3 2.1 Hz, 1H, Ar- H ), 5.39 (s, 2H, C H 2 ), 2.35 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 161.9, 152.4, 148.9, 146.5, 139.5, 135.3, 131.2, 129.1, 124.1, 123.3, 117.5, 115.0, 114.3, 105.8, 69.7, 20.0; m / z (ESI negative): 345.9 [M − H] − , m / z (ESI positive): 348.0 [M + H] + . 3-(benzyloxy)dibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 27 ) Compound 27 was obtained according to the general procedure earlier reported with benzyl bromide as starting material. Yield 51%; m.p. 232–235 °C; silica gel TLC Rf 0.69 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.20 (dd, J = 6.6 Hz, 2H, Ar- H ), 8.02 (d, J = 7.0 Hz, 1H, Ar- H ), 7.90 (dd, J = 7.0 Hz, 1H, Ar- H ), 7.67 (dd, J = 7.0 Hz, 1H, Ar- H ), 7.49 (d, J = 6.6 Hz, 2H, Ar- H ), 7.39 (m, 3H, Ar- H ), 7.24 (s, 1H, Ar- H ), 7.17 (d, J = 8.8 Hz, 1H, Ar- H ), 5.39 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 161.8, 151.1, 137.2, 135.7, 132.0, 131.1, 129.8, 129.5, 129.2, 129.0, 128.1, 126.1, 124.7, 115.4, 114.6, 106.7, 71.6; m / z (ESI negative): 336.9 [M − H] − , m / z (ESI positive): 339.0 [M + H] + . 3-((4-fluorobenzyl)oxy)dibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 28 ) Compound 28 was obtained according to the general procedure reported earlier with 4-fluorobenzyl bromide as starting material. Yield 49%; m.p. 268–269 °C; silica gel TLC Rf 0.81 (EtOAc/Hexane 50% v / v ): 8.26 (m, 2H, Ar- H ), 8.07 (d, J = 7.3 Hz, 1H, Ar- H ), 7.96 (m, 1H, Ar- H ), 7.72 (dd, J = 7.3 Hz, 1H, Ar- H ), 7.60 (m, 2H, Ar- H ), 7.30 (m, 2H, Ar- H ), 7.27 (d, J = 2.3 Hz, 1H, Ar- H ), 7.22 (dd, J = 8.4 2.3 Hz, 1H, Ar- H ), 5.26 (s, 2H, C H 2 ); δ H (400 MHz, DMSO-d 6 ); δ C (400 MHz, DMSO-d 6 ): 163.1 (d, J 1 = 186.4 Hz), 161.6, 151.1, 135.7, 133.4 (d, J 4 = 3.7 HZ), 132.0, 131.4 (d, J 3 = 8.6 Hz), 131.1, 129.8, 182.1, 126.1, 124.7, 116.5 (d, J 2 = 21.5 Hz), 115.4, 114.6, 106.7, 70.4; m / z (ESI negative): 354.9 [M − H] − , m / z (ESI positive): 357.1 [M + H] + . 3-((3-fluorobenzyl)oxy)dibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 29 ) Compound 29 was obtained according to the general procedure reported earlier with 3-fluorobenzyl bromide as starting material. Yield 76%; m.p. 249–252 °C; silica gel TLC Rf 0.77 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.27 (d, J = 2.5 Hz, 1H, Ar- H ), 8.25 (d, J = 3.3 Hz, 1H, Ar- H ), 8.07 (d, J = 7.7 Hz, 1H, Ar- H ), 7.95 (dd, J = 7.7 H, 1H, Ar- H ), 7.72 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.51 (q, J = 7.7 Hz, 1H, Ar- H ), 7.38 (m, 2H, Ar- H ), 7.30 (d, J = 2.5 Hz, 1H, Ar- H ), 7.23 (m, 2H, Ar- H ), 5.31 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 164.5 (d, J 1 = 243.9 Hz), 161.5, 151.2, 140.2 (d, J 3 = 7.3 HZ), 135.7, 132.0, 131.6 (d, J 3 = 8.6 Hz), 131.1, 129.9, 128.2, 126.2, 124.9 (d, J 4 = 2.6 Hz), 124.7, 116.0 (d, J 2 = 20.4 Hz), 115.7 (d, J 2 = 21.3 Hz), 115.4, 114.8, 106.8, 70.2; m / z (ESI negative): 354.9 [M − H] − , m / z (ESI positive): 357.0 [M + H] + . 3-((4-nitrobenzyl)oxy)dibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 30 ) Compound 30 was obtained according to the general procedure reported earlier with 4-nitrobenzyl bromide as starting material. Yield 72%; m.p. 264–266 °C; silica gel TLC Rf 0.43 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.28 (d, J = 8.1 Hz, 2H, Ar- H ), 8.23 (d, J = 8.8 Hz, 2H, Ar- H ), 8.03 (d, J = 7.7 Hz, 1H, Ar- H ), 7.91 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.76 (d, J = 8.1 H, 2H, Ar- H ), 7.68 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.27 (s, 1H, Ar- H ), 7.20 (d, J = 8.8 Hz, 1H, Ar- H ), 5.42 (s, 2H, C H 2 ); δ C (400 MHz, DMSO-d 6 ): 161.3, 151.1, 148.2, 145.1, 135.7, 131.9, 131.1, 130.0, 129.5, 128.3, 126.2, 124.8, 124.7, 115.4, 115.0, 106.8, 69.8; m / z (ESI negative): 382.0 [M − H] − , m / z (ESI positive): 383.9 [M + H] + . 3-((3-nitrobenzyl)oxy)dibenzo[c,e][1,2]oxathiine 6,6-dioxide ( 31 ) Compound 31 was obtained according to the general procedure reported earlier with 3-nitrobenzyl bromide as the starting material. Yield 70%; m.p. 248–251 °C; silica gel TLC Rf 0.56 (EtOAc/Hexane 50% v / v ): 8.36 (s, 1H, Ar- H ), 8.23 (s, J = 8.6 Hz, 3H, Ar- H ), 8.03 (d, J = 7.7 Hz, 1H, Ar- H ), 7.96 (d, J = 7.7 Hz, 1H, Ar- H ), 7.91 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.73 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.68 (dd, J = 7.7 Hz, 1H, Ar- H ), 7.29 (d, J = 2.3 Hz, 1H, Ar- H ), 7.22 (dd, J = 8.6 2.3 Hz, 1H, Ar- H ), 5.41 (s, 2H, C H 2 ); δ H (400 MHz, DMSO-d 6 ); δ C (400 MHz, DMSO-d 6 ): 161.3, 151.1, 148.9, 139.6, 135.7, 135.4, 131.9, 131.2, 131.1, 129.9, 128.3, 126.2, 124.7, 124.1, 123.4, 115.4, 114.9, 106.8, 69.7; m / z (ESI negative): 382.0 [M − H] − , m / z (ESI positive): 384.0 [M + H] + . General synthetic procedure for ethyl benzo[e][1,2]oxathiine-3-carboxylate 2,2-dioxide (34) and ethyl 7-methoxybenzo[e][1,2]oxathiine-3-carboxylate 2,2-dioxide (35) A solution of ethyl 2-(chlorosulfonyl)acetate (1.5 equiv.) in DCE (5 mL), prepared as reported by Liu et al., 1 was added dropwise in a sealed tube to a solution of 32 or 33 (1 g, 1 equiv.) and dry Py (2 equiv.) in DCE (3 mL). The resulting suspension was stirred at 90 °C for 4 h. Slush and HCl were added to the reaction mixture and the suspension was extracted in EtOAc (3 × 25 mL). The collected organic phase was washed with K 2 CO 3 s.s. (3 × 20 mL), dried with Na 2 SO 4 , filtered and evaporated to give product 34 and 35 as pale yellow powders in good yield and high purity. ethyl benzo[e][1,2]oxathiine-3-carboxylate 2,2-dioxide (34) Compound 34 was obtained according to the general procedure reported earlier with salicyl aldehyde ( 32 ) as starting material. Yield 43%; m.p. 111–113 °C; silica gel TLC Rf 0.54 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.59 (s, 1H, Ar- H ), 8.02 (d, J = 7.6 Hz, 1H, Ar- H ), 7.79 (dd, J = 7.6 Hz, 1H, Ar- H ), 7.59 (m, 2H, Ar- H ), 4.43 (q, J = 6.5 Hz, 2H, C H 2 ), 1.37 (dd, J = 6.5 Hz, 3H, C H 3 ); δ C (400 MHz, CDCl 3 -d 3 ): 159.7, 152.4, 142.2, 131.0, 126.3, 118.9, 118.8, 63.1, 14.1. ethyl 7-methoxybenzo[e][1,2]oxathiine-3-carboxylate 2,2-dioxide (35) Compound 35 was obtained according to the general procedure reported earlier with 4-methoxysalicylaldehyde ( 33 ) as starting material. Yield 41%; m.p. 167–169 °C; silica gel TLC Rf 0.49 (EtOAc/Hexane 50% v / v ); δ H (400 MHz, DMSO-d 6 ): 8.51 (s, 1H, Ar- H ), 7.94 (d, J = 8.6 Hz, 1H, Ar- H ), 7.22 (d, J = 2.2 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.6 2.2 Hz, 1H, Ar- H ), 4.40 (q, J = 6.5 Hz, 2H, C H 2 ), 3.95 (s, 3H, C H 3 ), 1.35 (dd, J = 6.5 Hz, 3H, C H 3 ); δ C (400 MHz, CDCl 3 -d 3 ): 164.8, 160.1, 154.4, 142.4, 132.4, 123.8, 113.4, 111.9, 103.8, 62.8, 56.1, 14.1. General synthetic procedure for benzo[e][1,2]oxathiine-3-carboxylic acid 2,2-dioxide (36) and 7-methoxybenzo[e][1,2]oxathiine-3-carboxylic acid 2,2-dioxide (37) NaOH 5M (7 equiv.) was added to a solution of appropriate ethyl esthers 34 or 35 (1g, 1 equiv.) in EtOH (20 mL), and the suspension was heated at reflux for 1h. The reaction mixuture was cooled and slushed, and HCl 6M were added to pH = 2. The resulting precipitate was filtered and washed with H 2 O to give us compounds 36 and 37 as a white powder in high yield and purity. benzo[e][1,2]oxathiine-3-carboxylic acid 2,2-dioxide (36) Compound 36 was obtained according to the general procedure reported earlier with 34 as starting material. Yield 81%; m.p. 105–107 °C; silica gel TLC Rf 0.04 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 14.57 (s, 1H, exchange with D 2 O, COO H ), 8.50 (s, 1H, Ar- H ), 7.98 (d, J = 7.4 Hz, 1H, Ar- H ), 7.71 (dd, J = 7.4 Hz, 1H, Ar- H ), 7.55 (m, 2H, Ar- H ); δ C (400 MHz, CDCl 3 -d 3 ): 169.1, 159.4, 142.1, 132.5, 126.7, 118.8, 118.5. 7-methoxybenzo[e][1,2]oxathiine-3-carboxylic acid 2,2-dioxide (37) Compound 37 was obtained according to the general procedure reported earlier with 35 as starting material. Yield 73%; m.p. 150–151 °C; silica gel TLC Rf 0.01 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 14.31 (s, 1H, exchange with D 2 O, COO H ), 8.41 (s, 1H, Ar- H ), 7.89 (d, J = 8 Hz, 1H, Ar- H ), 7.18 (d, J = 2.1 Hz, 1H, Ar- H ), 7.10 (dd, J = 8 2.1 Hz, 1H, Ar- H ), 3.94 (s, 3H, C H 3 ); δ C (400 MHz, CDCl 3 -d 3 ): 170.0, 157.6, 154.7, 140.6, 132.0, 123.1, 113.4, 111.8, 103.9, 56.0. General synthetic procedure for products 38–47 PyBOP (1.2 equiv.) and appropriate aniline (1.2 equiv.) were added at 0 °C to a solution of 36 or 37 (0.2 g, 1 equiv.) in dry DMF (2 mL) under nitrogen atmosphere. DIPEA (3 equiv.) were added dropwise at 0 °C and the resulting solution was stirred o.n. at RT. The reaction mixture was quenched with slush and HCl 6M and the H 2 O-phase was extracted in EtOAc (3 × 30 mL). The collected organic phases were washed with HCl 1M (3 × 20 mL), NaHCO 3 (2 × 20 mL) and brine (3 × 20 mL), then the organic phase was dried with Na 2 SO 4 , filtered and evaporated under vacuum to obtain. All the products were purified by silica gel chromathography (MeOH/DCM 0.5% v / v ). N -phenylbenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (38) Compound 38 was obtained according to the general procedure reported earlier with aniline as starting material. Yield 60%; m.p. 192–195 °C; silica gel TLC Rf 0.82 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.83 (s, 1H, exchange with D 2 O, CON H ), 8.35 (s, 1H, Ar- H ), 7.92 (d, J = 8.0 Hz, 1H, Ar- H ), 7.74 (m, 3H, Ar- H ), 7.58 (m, 2H, Ar- H ), 7.44 (dd, J = 7.3 Hz, 2H, Ar- H ), 7.21 (dd, J = 7.3 Hz, 1H, Ar- H ); δ C (400 MHz, DMSO-d 6 ): 159.0, 152.1, 139.1, 138.5, 135.2, 132.4, 132.3, 130.0, 127.9, 125.6, 121.1, 120.0, 119.8; m / z (ESI negative): 299.9 [M − H] − , m / z (ESI positive): 302.0 [M + H] + . N -(4-bromophenyl)benzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (39) Compound 39 was obtained according to the general procedure reported earlier with 4-bromoaniline as starting material. Yield 21%; m.p. 249–252 °C; silica gel TLC Rf 0.88 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.96 (s, 1H, exchange with D 2 O, CON H) , 8.36 (s, 1H, Ar- H ), 7.92 (dd, J = 7.7 1.4 Hz, 1H, Ar- H ), 7.76 (ddd, J = 7.7 1.4 Hz, 1H, Ar- H ), 7.69 (d, J = 8.8 Hz, 2H Ar- H ), 7.62 (d, J = 8.8 Hz, 2H, Ar- H ), 7.58 (m, 2H, Ar- H ); δ C (400 MHz, DMSO-d 6 ): 159.1, 152.1, 138.9, 138.5, 135.4, 132.8, 132.3, 132.2, 128.0, 123.0, 119.9, 119.8, 117.4; m / z (ESI negative): 379.9 [M − H] − , m / z (ESI positive): 381.9 [M + H] + . N -(3-bromophenyl)benzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (40) Compound 40 was obtained according to the general procedure reported earlier with 3-bromoaniline as starting material. Yield 38%; m.p. 228–229 °C; silica gel TLC Rf 0.90 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.96 (s, 1H, exchange with D 2 O, CON H) , 8.38 (s, 1H, Ar- H ), 8.01 (s, 1H, Ar- H ), 7.91 (dd, J = 7.9 1.7 Hz, 1H Ar- H ), 7.77 (ddd, J = 7.9 1.7 Hz, 1H, Ar- H ), 7.67 (m, 1H, Ar- H ), 7.58 (m, 2H, Ar- H ), 7.41 (d, J = 5.5 Hz, 2H, Ar- H ); δ C (400 MHz, DMSO-d 6 ): 159.2, 152.2, 140.7, 139.0, 135.4, 132.3, 132.1, 132.0, 128.2, 128.0, 123.5, 122.6, 119.9, 119.8, 118.2; m / z (ESI negative): 379.9 [M − H] − , m / z (ESI positive): 381.9 [M + H] + . N -(4-methoxyphenyl)benzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (41) Compound 41 was obtained according to the general procedure reported earlier with 4-methoxyaniline as starting material. Yield 37%; m.p. 231–233 °C; silica gel TLC Rf 0.82 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.70 (s, 1H, exchange with D 2 O, CON H) , 8.30 (s, 1H, Ar- H ), 7.90 (dd, J = 7.6 1.8 Hz, 1H, Ar- H ), 7.75 (ddd, J = 7.6 1.8 Hz, 1H, Ar- H ), 7.63 (d, J = 9.0 Hz, 2H, Ar- H ), 7.56 (m, 2H, Ar- H ), 7.00 (d, J = 9.0 Hz, 2H, Ar- H ), 3.80 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 158.6, 157.1, 152.1, 138.1, 135.1, 132.6, 132.2, 132.1, 127.9, 122.6, 120.0, 119.8, 115.1, 56.3; m / z (ESI negative): 329.9 [M − H] − , m / z (ESI positive): 332.0 [M + H] + . N -(3-methoxyphenyl)benzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (42) Compound 42 was obtained according to the general procedure reported earlier with 3-methoxyaniline as starting material. Yield 31%; m.p. 219–221 °C; silica gel TLC Rf 0.94 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.79 (s, 1H, exchange with D 2 O, CON H) , 8.34 (s, 1H, Ar- H ), 7.91 (dd, J = 7.5 1.3 Hz, 1H, Ar- H ), 7.76 (ddd, J = 7.5 1.3 Hz, 1H, Ar- H ), 7.57 (m, 2H, Ar- H ), 7.33 (m, 3H, Ar- H ), 6.79 (dd, J = 8.4 1.8 Hz, 1H, Ar- H ), 3.81 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 160.6, 159.0, 152.1, 140.3, 138.6, 135.3, 132.4, 132.3, 130.8, 128.0, 119.9, 119.8, 113.3, 111.1, 106.7, 56.1; m / z (ESI negative): 329.9 [M − H] − , m / z (ESI positive): 332.0 [M + H] + . 7-methoxy-N-phenylbenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (43) Compound 43 was obtained according to the general procedure reported earlier with aniline as starting material. Yield 50%; m.p. 241–244 °C; silica gel TLC Rf 0.76 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.71 (s, 1H, exchange with D 2 O, CON H) , 8.29 (s, 1H, Ar- H ), 7.83 (d, J = 8.4 Hz, 1H, Ar- H ), 7.71 (d, J = 7.1 Hz, 2H, Ar- H ), 7.43 (dd, J = 7.1 Hz, 2H, Ar- H ), 7.17 (m, 3H, Ar- H ), 3.94 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 164.9, 159.3, 154.1, 139.3, 138.9, 133.6, 129.9, 129.0, 125.4, 121.0, 114.7, 112.7, 105.1, 57.4; m / z (ESI negative): 329.9 [M − H] − , m / z (ESI positive): 332.0 [M + H] + . N -(4-bromophenyl)-7methoxybenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (44) Compound 44 was obtained according to the general procedure reported earlier with 4-bromoaniline as starting material. Yield 38%; m.p. 272–275 °C; silica gel TLC Rf 0.92 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.83 (s, 1H, exchange with D 2 O, CON H) , 8.31 (s, 1H, Ar- H ), 7.83 (d, J = 8.4 Hz, 1H, Ar- H ), 7.68 (d, J = 9.1 Hz, 2H, Ar- H ), 7.61 (d, J = 9.1 Hz, 2H, Ar- H ), 7.22 (d, J = 2.2 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.4 2.2 Hz, 1H, Ar- H ), 3.94 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 165.0, 159.4, 154.1, 139.2, 138.7, 133.7, 132.8, 128.8, 122.9, 117.2, 114.8, 112.7, 105.2, 57.4; m / z (ESI negative): 407.9 [M − H] − , m / z (ESI positive): 411.9 [M + H] + . N -(3-bromophenyl)-7-methoxybenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (45) Compound 45 was obtained according to the general procedure reported earlier with 3-bromoaniline as starting material. Yield 32%; m.p. 218–220 °C; silica gel TLC Rf 0.87 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.80 (s, 1H, exchange with D 2 O, CON H) , 8.26 (s, 1H, Ar- H ), 7.96 (s, 1H, Ar- H ), 7.78 (d, J = 8.3 Hz, 1H, Ar- H ), 7.62 (d, J = 4.5 Hz, 1H, Ar- H ), 7.35 (m, 2H, Ar- H ), 7.18 (d, J = 2.2 Hz, 1H, Ar- H ), 7.08 (dd, J = 8.3 2.2 Hz, 1H, Ar- H ), 3.90 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 165.1, 159.6, 154.1, 140.8, 139.4, 133.7, 132.0, 128.6, 128.0, 123.4, 122.6, 119.8, 114.8, 112.6, 105.2, 57.4; m / z (ESI negative): 409.9 [M − H] − , m / z (ESI positive): 411.9 [M + H] + . N -(4-methoxyphenyl)-7-methoxybenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (46) Compound 46 was obtained according to the general procedure reported earlier with 4-methoxyaniline as starting material. Yield 58%; m.p. 238–240 °C; silica gel TLC Rf 0.88 (MeOH/DCM 5% v / v ): 10.57 (s, 1H, exchange with D 2 O, CON H) , 8.24 (s, 1H, Ar- H ), 7.82 (d, J = 8.2 Hz, 1H, Ar- H ), 7.62 (d, J = 8.8 Hz, 2H, Ar- H ), 7.21 (d, J = 2.2 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.2 2.2 Hz, 1H, Ar- H ), 6.99 (d, J = 8.8 Hz, 2H, Ar- H ), 3.97 (s, 3H, C H 3 ), 3.82 (s, 3H, C H 3 ); δ H (400 MHz, DMSO-d 6 ); δ C (400 MHz, DMSO-d 6 ): 164.8, 158.9, 157.1, 154.0, 138.4, 133.5, 132.2, 129.2, 122.6, 115.1, 114.7, 112.8, 105.1, 57.4, 56.3; m / z (ESI negative): 359.9 [M − H] − , m / z (ESI positive): 362.0 [M + H] + . N -(3-methoxyphenyl)-7-methoxybenzo[e][1,2]oxathiine-3-carboxamide 2,2-dioxide (47) Compound 47 was obtained according to the general procedure reported earlier with 3-methoxyaniline as starting material. Yield 41%; m.p. 222–225 °C; silica gel TLC Rf 0.79 (MeOH/DCM 5% v / v ); δ H (400 MHz, DMSO-d 6 ): 10.69 (s, 1H, exchange with D 2 O, CON H) , 8.27 (s, 1H, Ar- H ), 7.37 (dd, J = 2.0 Hz, 1H, Ar- H ), 7.32 (dd, J = 8.0 Hz, 1H, Ar- H ), 7.27 (d, J = 8.0 Hz, 1H, Ar- H ), 7.22 (d, J = 2.1 Hz, 1H, Ar- H ), 7.12 (dd, J = 8.6 2.1 Hz, 1H, Ar- H ), 6.77 (dd, J = 8.0 2.0 Hz, 1H, Ar- H ), 3.94 (s, 3H, C H 3 ), 3.80 (s, 3H, C H 3 ); δ C (400 MHz, DMSO-d 6 ): 164.9, 160.6, 159.3, 154.1, 144.6, 140.4, 138.9, 133.6, 130.7, 129.0, 114.7, 113.2, 111.0, 106.7, 105.1, 57.4, 56.1; m / z (ESI negative): 359.9 [M − H] − , m / z (ESI positive): 362.0 [M + H] + . 3.2. Carbonic Anhydrase Inhibition An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed CO 2 hydration activity [ 44 ]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10–100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6h at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation and represent the mean from at least three different determinations. The enzyme concentrations were in the range 5–16 nM. All CA isoforms were recombinant ones obtained in-house [ 47 , 48 ]. 4. Conclusions We report here a series of 7-substituted and 3-substituted sulfocoumarins, obtained by cyclization of 2-hydroxy-4-methoxybenzaldehyde (7), 2′-hydroxy-4′-methoxyacetophenon (8), 2-bromobenzenesulfonyl chloride (23), salicylaldehyde (32) or 4-methoxysalicylaldehyde (33) and possessing different substituents in position 3 or 7 of the heterocyclic ring. 7-Substituted sulfocoumarins resulted in being good inhibitors of hCA IX and XII, whilst 3-substituted sulfocoumarins showed a worsening in terms of inhibition potency on this isoform. A common feature of all the synthesized products was the absence of inhibition on hCA I and II. The most potent products belong to Type 1 group, probably due to the absence of the bulky group on the heterocyclic ring; particularly, sulfocoumarin 15 showed a nanomolar inhibition on hCA IX and XII and turned out to be the most potent inhibitor achieved (hCA IX: K I = 22.9 nM; hCA XII: K I = 19.2 nM). The structure-activity relationship for this class of CAIs has been expanded considering the synthesis of 3-substituted sulfocoumarines for the first time. The observed isoform-selective inhibition displayed here may be considered to be of interest for various biomedical applications. Author Contributions Synthesis, characterization, and in vitro assays, enzyme preparation, C.C. and S.G.; original draft preparation, S.G.; design and supervision, A.N.; funding, C.T.S.; writing—review and editing, C.T.S. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Italian Ministry for University and Research (MIUR), grant PRIN: prot. 2017XYBP2R and Fondazione Cassa di Risparmio di Firenze grant CRF2020.1395. (CTS). Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Compounds or related data available directly from the authors. Conflicts of Interest The authors do not declare conflict of interest. References Supuran, C.T. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008 , 7 , 168–181. [ Google Scholar ] [ CrossRef ] [ PubMed ] Supuran, C.T.; Capasso, C. An overview of the bacterial carbonic anhydrases. Metabolites 2017 , 7 , 56. [ Google Scholar ] Supuran, C.T. Structure and function of carbonic anhydrases. Biochem. J. 2016 , 473 , 2023–2032. [ Google Scholar ] [ CrossRef ] [ PubMed ] Neri, D.; Supuran, C.T. 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Coumarins 1 and 2, and sulfocoumarin 3 and their CA-mediated hydrolysis to the CAI active species 4 , 5 and 6 , respectively. Figure 2. A ribbon view of the active site of hCA II in adduct with ( A ) Compound 4 (cyan, PDB 5BNL), derived from the CA-mediated hydrolysis of coumarin 1 ; ( B ) Compound 5 (magenta, PDB 3F8E), derived from the CA-mediated hydrolysis of coumarin 2 . ( C ) Compound 6 (green, PDB 4BCW), derived from the CA-mediated hydrolysis of sulfocoumarin 3 . The Zn(II) is shown as a grey sphere that is bound to the protein His ligands (labels not shown). Water molecules are represented as red spheres. H-bonds are represented as black dashed lines. Figure 2. A ribbon view of the active site of hCA II in adduct with ( A ) Compound 4 (cyan, PDB 5BNL), derived from the CA-mediated hydrolysis of coumarin 1 ; ( B ) Compound 5 (magenta, PDB 3F8E), derived from the CA-mediated hydrolysis of coumarin 2 . ( C ) Compound 6 (green, PDB 4BCW), derived from the CA-mediated hydrolysis of sulfocoumarin 3 . The Zn(II) is shown as a grey sphere that is bound to the protein His ligands (labels not shown). Water molecules are represented as red spheres. H-bonds are represented as black dashed lines. Figure 3. The structure of SLC-0111, a specific hCA IX and XII inhibitor studied in phase Ib/II clinical trials for the treatment of hypoxic tumors. Figure 3. The structure of SLC-0111, a specific hCA IX and XII inhibitor studied in phase Ib/II clinical trials for the treatment of hypoxic tumors. Scheme 1. A synthetic approach for obtaining products 13 – 22 . Reagents and conditions: (a) Mesylchloride, Et 3 N, dry DCM, 0 °C to RT, 2 h; (b) DBU, dry DCM, 0 °C to RT, o.n.; (c) dry Py, dry DCM, 0 °C to RT, o.n.; (d) BBr 3 , dry DCM, − 10 °C to RT, o.n.; (e) appropriate benzyl bromide, K 2 CO 3 , dry DMF, RT, o.n. Scheme 1. A synthetic approach for obtaining products 13 – 22 . Reagents and conditions: (a) Mesylchloride, Et 3 N, dry DCM, 0 °C to RT, 2 h; (b) DBU, dry DCM, 0 °C to RT, o.n.; (c) dry Py, dry DCM, 0 °C to RT, o.n.; (d) BBr 3 , dry DCM, − 10 °C to RT, o.n.; (e) appropriate benzyl bromide, K 2 CO 3 , dry DMF, RT, o.n. Scheme 2. A synthetic approach to obtain products 27 – 31 . Reagents and conditions: (a) 3-methoxyphenol, Et 3 N, dry DCM, 0 °C to RT, 3 h; (b) KOAc, Pd(OAc) 2 , dry DMA, 150 °C, 4 h; (c) BBr 3 , dry DCM, − 10 °C to RT, o.n.; (d) appropriate benzyl bromide, K 2 CO 3 , dry DMF, RT, o.n. Scheme 2. A synthetic approach to obtain products 27 – 31 . Reagents and conditions: (a) 3-methoxyphenol, Et 3 N, dry DCM, 0 °C to RT, 3 h; (b) KOAc, Pd(OAc) 2 , dry DMA, 150 °C, 4 h; (c) BBr 3 , dry DCM, − 10 °C to RT, o.n.; (d) appropriate benzyl bromide, K 2 CO 3 , dry DMF, RT, o.n. Scheme 3. A synthetic approach to obtain products 38–47. Reagents and conditions: (a) ethyl 2-chlorosulfonyl acetate, dry Py, DCE, 90 °C, 2 h; (b) NaOH 5M, EtOH, reflux, 30′; (c) appropriate aniline, PyBOP, DIPEA, dry DMF, 0 °C to RT, o.n. Scheme 3. A synthetic approach to obtain products 38–47. Reagents and conditions: (a) ethyl 2-chlorosulfonyl acetate, dry Py, DCE, 90 °C, 2 h; (b) NaOH 5M, EtOH, reflux, 30′; (c) appropriate aniline, PyBOP, DIPEA, dry DMF, 0 °C to RT, o.n. Table 1. Inhibition data of hCA isoforms I, II, IX and XII with sulfocoumarins 13 – 22 , 27 – 31 , 38 – 47 and the standard sulfonamide inhibitor AAZ by a stopped-flow CO 2 hydrase assay [ 44 ]. Table 1. Inhibition data of hCA isoforms I, II, IX and XII with sulfocoumarins 13 – 22 , 27 – 31 , 38 – 47 and the standard sulfonamide inhibitor AAZ by a stopped-flow CO 2 hydrase assay [ 44 ]. Cmpd K I (nM) a,b R 1 R 2 CA I CA II CA IX CA XII 9 H OCH 3 >100,000 15,360 74.9 61.4 10 CH 3 OCH 3 >100,000 8814 97.2 73.9 11 H H 16,538 2896 55.8 42.5 12 CH 3 H 9961 3857 81.7 55.0 13 H H >100,000 >100,000 55.6 28.3 14 H 4-F >100,000 >100,000 22.9 19.2 15 H 3-F >100,000 >100,000 28.4 46.0 16 H 4-NO 2 >100,000 >100,000 69.8 94.7 17 H 3-NO 2 >100,000 >100,000 40.2 77.5 18 CH 3 H >100,000 >100,000 49.8 8.6 19 CH 3 4-F >100,000 >100,000 14.3 41.7 20 CH 3 3-F >100,000 >100,000 33.2 25.7 21 CH 3 4-NO 2 >100,000 >100,000 89.7 100.8 22 CH 3 3-NO 2 >100,000 >100,000 61.4 136.0 25 OCH 3 - >100,000 >100,000 1286 965.8 26 H - 42650 8997 558.9 414.2 27 H - >100,000 >100,000 680.5 910.7 28 4-F - >100,000 >100,000 754.9 653.8 29 3-F - >100,000 >100,000 615.1 566.4 30 4-NO 2 - >100,000 >100,000 964.7 1056 31 3-NO 2 - >100,000 >100,000 898.1 1369 34 H CH 2 CH 3 >100,000 >100,000 1631 2594 35 OCH 3 CH 2 CH 3 >100,000 >100,000 1085 1528 36 H H 4625 996.5 499.7 563.7 37 OCH 3 H 7139 728.1 853.7 408.2 38 H H >100,000 >100,000 3288 2301 39 H 4-Br >100,000 >100,000 34,350 58,980 40 H 3-Br >100,000 >100,000 40,840 78,320 41 H 4-OCH 3 >100,000 >100,000 8231 8550 42 H 3-OCH 3 >100,000 >100,000 5424 3380 43 OCH 3 H >100,000 >100,000 2429 1594 44 OCH 3 4-Br >100,000 >100,000 51,460 42,520 45 OCH 3 3-Br >100,000 >100,000 64,380 76,540 46 OCH 3 4-OCH 3 >100,000 >100,000 4367 6547 47 CH 3 3-OCH 3 >100,000 >100,000 6558 2469 AAZ - - 250 12.0 25.0 5.7 a . Mean from 3 different assays by a stopped flow technique (errors in the range of 5−10% of the reported values); b . 6 h incubation. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). MDPI and ACS Style Giovannuzzi, S.; Capasso, C.; Nocentini, A.; Supuran, C.T. Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII. Molecules 2022, 27, 4076. https://doi.org/10.3390/molecules27134076 Giovannuzzi S, Capasso C, Nocentini A, Supuran CT. Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII. Molecules. 2022; 27(13):4076. https://doi.org/10.3390/molecules27134076 Giovannuzzi, Simone, Clemente Capasso, Alessio Nocentini, and Claudiu T. Supuran. 2022. "Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII" Molecules27, no. 13: 4076. https://doi.org/10.3390/molecules27134076	https://www.mdpi.com/1420-3049/27/13/4076/xml
The Secret to Happiness? Gratitude - How to Practice Gratitude By practicing gratitude, you can make yourself thankful for the simplest things. And that can make a big difference in your life. The Secret to Happiness? Gratitude. Just ask yourself this simple question. sbayram // Getty Images Something amazing happened to me this morning: I woke up. I don’t mean I suddenly experienced a religious conversion or became “woke” as in socially-conscious. I mean I quite literally, and simply, opened my eyes. Through some miracle and mystery of consciousness I woke up to the reality of my life, ready for another day. Another day to drink coffee, text a friend, listen to music, stare into space, have sex, comb my hair, eat bacon, and check Twitter. Another day to call my Grandpa, drink a beer, read a book, sneeze, forget to brush my teeth, receive a compliment, and worry about what the hell I’m going to write about. But it’s not just me. Youwoke up today, too. This is a big deal. Especially when you realize that there will come a day when you will NEVER WAKE UP AGAIN. Consider this: yesterday 150,000 people from all around the world died from all sorts of diseases, accidents, and acts of violence. Today, another 150,000 people will die. Tomorrow another 150,000. And the day after that. And the day after that. And the day after...you get the idea. KatarzynaBialasiewicz // Getty Images Over the course of a single calendar year, 55.3 million people will take their last breath. By the time you finish reading this sentence, ten people will have died. Hopefully you weren’t one of them……. Still here? OK, moving on. It’s worth calling attention to the fact of death needn’t be nihilistic or depressing. In fact, with the right mindset, becoming aware of your own mortality can have the opposite effect: It can flood your entire existence with the feeling of gratitude. Gratitude Is Good For Us Researcher Robert Emmons, considered by many to be the world’s leading scientific expert on gratitude, believes that gratitude has two components. “First,” he writes, “it’s an affirmation of goodness. We affirm there are good things in the world, gifts and benefits we’ve received. Second, we recognize the sources of this goodness as being outside ourselves.” AleksandarNakic // Getty Images In other words, we didn’t necessarily create the circumstances that gave rise to the goodness. Instead, as Emmons puts it, “we acknowledge that other people—or even higher powers, if you’re of a spiritual mindset—gave us many gifts, big and small, to help us achieve the goodness in our lives. There’s now a mountain of science showing that the practice of gratitude is good for us. In one seminal 2003 study, Emmons and fellow researcher Michael McCullough split people into three groups. Participants in Group 1 were instructed to think back over the past week and write down five things they were grateful for. Participants in Group 2 were asked to write down five hassles or annoying things they’d experienced. And finally, participants in Group 3, the control group, were asked to down five events that had “affected” them in the past week, whether positive, negative, or neutral. BraunS // Getty Images After 10 weeks of this practice, results showed that participants in the gratitude group felt better about their life, were more excited about the coming week, and experienced fewer symptoms of physical illness. Since then, dozens more studies have been completed. Gratitude has now been shown to decrease depression, increase empathy, improve self-esteem, and build mental resilience. We know this, of course. But practicing gratitude can be difficult. How are we supposed to feel grateful when we get fired from our job or get into a fight with our partner? How are we supposed to feel gratitude when life seems to be a never-ending source of small disappointments and failures? Most people feel as if they need to wait for something good to happen in order to feel grateful. Luckily (See? I’m doing it right now), this is not the case. Instead, you can reliably createthe feeling of gratitude in two powerful ways: Think about all the bad stuff that hasn’t happened to you Recognize the beauty and complexity of simple things you normally take for granted IT COULD BE WORSE! This is a tip I got from Sam Harris, a neuroscientist and philosopher. In his extremely practical meditation app, Waking Up, Harris instructs us to periodically “manufacture a feeling of gratitude by simply contemplating all the bad stuff that hasn’t happened to you.” LittleBee80 // Getty Images Let’s say you’re stuck in traffic and late for an important meeting. In this situation, most of us feel angry and stressed out. You curse the cars in front of you. You beat yourself up for not leaving earlier. You worry about the consequences of your being late. But as Harris instructs, there is another move open to us: You can stop freaking out and think about how many people around the world would happily trade places with you in order to escape the current reality of their dangerous, less lucky lives. People caught in war zones, migrants walking hundreds of miles in blistering heat...these are realities that exist in this moment, and you are not in them. You are simply sitting in traffic, fully clothed, fed, and air-conditioned, with no real worries. As Harris reminds us, “There are at least a billion people on earth at this moment who would consider their prayers answered if they could only live as you do now.” Kosamtu // Getty Images Recognizing this fact can transform an ordinary feeling of annoyance into a deep sense of gratitude for your life—and compassion for the people who find themselves in more unfortunate situations. Marvel at the Simple Things In his new book, Thanks A Thousand, author A.J. Jacobs opens with: "This marvel I see before me is the result of thousands of human beings collaborating across dozens of countries. It took the combined labor of artists, chemists, politicians, mechanics, biologists, miners, packagers, smugglers, and goatherds. It required airplanes, boats, trucks, motorcycles, vans, pallets, and shoulders.” The object of his amazement? A cup of coffee. In his book, Jacobs is on a mission to thank every single person involved in producing his morning cup of coffee, from the barista who served it to him to the farmers in Colombia who grew the coffee (and everyone in between). Marty Mathieu // Getty Images His point is profound: Even the smallest things we normally take for granted are incredibly rich, complex, and amazing. Allowing ourselves to be in awe of simple things can increase our feeling of gratitude and wellbeing. You can do this with virtually everything you encounter during your day. For instance, what happens when you charge your phone? What’s actually going on? Well, dozens or hundreds of miles away at a power plant, an energy source powers a generator which produces an electrical current. This current goes through giant transformers to increase the voltage. Through a sprawling network of power lines, the electrical charge arrives at your local substation where the voltage is lowered and sent to smaller power lines in your neighborhood. The current enters a smaller transformer outside your house, which then goes into your service panel where breakers and fuses protect the wires in your house from getting overloaded. Then the electricity travels through wires in your home to an outlet you use to charge your phone. (Leaving aside all the miraculous things your phone does.) All that just so you read this article, watch stupid cat videos, swipe right on Tinder, and avoid all the work you’re supposed to be doing right now. More Gratitude = A Better Life Knowing how to manufacture a feeling of gratitude is a skill that really can make every aspect of your life better. The good news is you don’t have to wait for anything good to happen. “Unless you’re living the worst possible life,” says Harris, “it should be easy to find something for which you’re grateful.” Whether you do that by thinking of all the bad stuff that hasn’t happened to you, or by recognizing the beauty of simple things (like the fact you have eyes to read this), you can feel a deep sense of wellbeing and ease in every moment of your life. It’s like Drake, our honorary pop-culture poet laureate, reminds us: What a time to be alive. Advertisement - Continue Reading Below	https://www.menshealth.com/health/a25562952/gratitude-secret-to-happiness/
Sensors \| Free Full-Text \| A GPS-Referenced Wavelength Standard for High-Precision Displacement Interferometry at & lambda; = 633 nm Since the turn of the millennium, the development and commercial availability of optical frequency combs has led to a steadily increase of worldwide installed frequency combs and a growing interest in using them for industrial-related metrology applications. Especially, GPS-referenced frequency combs often serve as a & ldquo;self-calibrating& rdquo; length standard for laser wavelength calibration in many national metrology institutes with uncertainties better than u = 1 & times; 10& minus;11. In this contribution, the application of a He-Ne laser source permanently disciplined to a GPS-referenced frequency comb for the interferometric measurements in a nanopositioning machine with a measuring volume of 200 mm & times; 200 mm & times; 25 mm (NPMM-200) is discussed. For this purpose, the frequency stability of the GPS-referenced comb is characterized by heterodyning with a diode laser referenced to an ultrastable cavity. Based on this comparison, an uncertainty of u = 9.2 & times; 10& minus;12 (& tau; = 8 s, k = 2) for the GPS-referenced comb has been obtained. By stabilizing a tunable He-Ne source to a single comb line, the long-term frequency stability of the comb is transferred onto our gas lasers increasing their long-term stability by three orders of magnitude. Second, short-term fluctuations-related length measurement errors were reduced to a value that falls below the nominal resolving capabilities of our interferometers (& Delta;L/L = 2.9 & times; 10& minus;11). Both measures make the influence of frequency distortions on the interferometric length measurement within the NPMM-200 negligible. Furthermore, this approach establishes a permanent link of interferometric length measurements to an atomic clock. by Ulrike Blumröder 1,* , Paul Köchert 2 , Thomas Fröhlich 1 , Thomas Kissinger 1 , Ingo Ortlepp 1 , Jens Flügge 2 , Harald Bosse 2 and Eberhard Manske 1 1 Institute of Process Measurement and Sensor Technology, Technische Universität Ilmenau, 98693 Ilmenau, Germany 2 Precision Engineering Division, Physikalisch Technische Bundesanstalt (PTB), 38116 Braunschweig, Germany * Author to whom correspondence should be addressed. Sensors 2023 , 23 (3), 1734; https://doi.org/10.3390/s23031734 Received: 17 November 2022 / Revised: 14 December 2022 / Accepted: 14 January 2023 / Published: 3 February 2023 (This article belongs to the Special Issue Precision Metrology Using Ultrashort Pulse Laser and Optical Frequency Comb ) Versions Notes Abstract Since the turn of the millennium, the development and commercial availability of optical frequency combs has led to a steadily increase of worldwide installed frequency combs and a growing interest in using them for industrial-related metrology applications. Especially, GPS-referenced frequency combs often serve as a “self-calibrating” length standard for laser wavelength calibration in many national metrology institutes with uncertainties better than u = 1 × 10 −11 . In this contribution, the application of a He-Ne laser source permanently disciplined to a GPS-referenced frequency comb for the interferometric measurements in a nanopositioning machine with a measuring volume of 200 mm × 200 mm × 25 mm (NPMM-200) is discussed. For this purpose, the frequency stability of the GPS-referenced comb is characterized by heterodyning with a diode laser referenced to an ultrastable cavity. Based on this comparison, an uncertainty of u = 9.2 × 10 −12 ( τ = 8 s, k = 2) for the GPS-referenced comb has been obtained. By stabilizing a tunable He-Ne source to a single comb line, the long-term frequency stability of the comb is transferred onto our gas lasers increasing their long-term stability by three orders of magnitude. Second, short-term fluctuations-related length measurement errors were reduced to a value that falls below the nominal resolving capabilities of our interferometers (Δ L / L = 2.9 × 10 −11 ). Both measures make the influence of frequency distortions on the interferometric length measurement within the NPMM-200 negligible. Furthermore, this approach establishes a permanent link of interferometric length measurements to an atomic clock. Keywords: GPS-disciplined oscillator ; optical frequency comb ; traceability ; ultrastable laser ; displacement interferometry ; nanopositioning and nanomeasuring machine (NPMM) 1. Introduction The demand for precise frequency measurements in the optical domain, the coalescence of decades of research and development in the field of pulsed laser systems, laser spectroscopy and nonlinear material interaction, as well as new findings on pulse propagation in micro structured optical fibers have led to the rise of the optical frequency comb technology at the turn of the millennium [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. An optical frequency υ n within the spectrum of a mode-locked pulse laser is defined by its respective mode number n , the repetition rate f Rep , and carrier envelope offset (CEO) frequency f CEO [ 8 ]: υ n = n · f Rep ± f CEO , (1) Both comb parameters f Rep and f CEO are usually in the rf domain, thus phase locking them to an atomic clock provides a direct link of an optical frequency to a primary frequency standard and consequently, the SI meter definition. In this way, ultraprecise, traceable optical frequency measurements can now be performed with unprecedented small experimental effort and cost. Within the revision of the practical realization of the definition of the meter by the Comité international des poids et mesures (CIPM) in 2002, it was considered “… that new femtosecond comb techniques have clear significance for relating the frequency of high-stability optical frequency standards realizing the SI second, that these techniques represent a convenient measurement technique for providing traceability to the International System of Units (SI) and that comb technology also can provide frequency sources as well as a measurement technique.” [ 9 ] (p. 182), [ 10 ] (p. 104). Furthermore, the CIPM recognized “comb techniques as timely and appropriate” [ 9 ] (p. 182), [ 10 ] (p. 104) and urged ”national metrology institutes and other laboratories to pursue the comb technique to the highest level of accuracy achievable and also to seek simplicity so as to encourage widespread application” [ 9 ] (p. 182), [ 10 ] (p. 104). Therefore, frequency combs quickly started to complement and replace common frequency standards, such as the iodine-stabilized He-Ne laser in national metrology institutes [ 11 , 12 ] and spurred research in a wide range of high-precision metrology applications [ 2 , 4 ]. Especially GPS-referenced frequency combs provide a cost-effective alternative to an atomic clock and can serve as a “self-calibrating” wavelength standard for laser wavelength calibration where an uncertainty of 10 −12 appears to be sufficient [ 13 , 14 ]. Their unique features also turned them into a versatile tool for precise interferometric distance measurements that are playing a crucial role for the advance of precision engineering and nanotechnology [ 15 ]. Especially for applications in nanotechnology, length metrology has to face tremendous challenges due to increasing demands on measurement range, accuracy, traverse speed, and traceability (see e.g., [ 16 ]). Herein, the combination of state-of-the-art interferometric methods with optical frequency combs has put the term “high-precision” to the next level and created new possibilities for absolute and incremental interferometric length measurements. Absolute distance measurements based on multiwavelength and dispersive interferometry benefit from the broad bandwidth of frequency combs and the high temporal coherence of individual comb lines as well as their traceable, absolutely known frequencies when a comb is referenced to an atomic clock [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. For applications in absolute distance measurements, one option is to use the comb directly in a Michelson interferometer configuration with a spectrally resolved detection scheme [ 17 , 18 , 19 ]. Therein, the application of a high-resolution spectrometer has made a spectral resolution to the level of individual comb modes for a comb repetition rate of 1 GHz possible and a combination of spectral interferometry and multiwavelength, homodyne interferometry was applied to determine distances within an unambiguity range of 15 cm and a standard deviation of 28 nm compared to conventional homodyne displacement interferometry [ 19 ]. Lately, this method has been evaluated with an incremental He-Ne laser interferometer demonstrating an agreement in the micrometer range over distances up to 50 m [ 20 ]. A second option is to utilize individual modes of the comb spectrum as a traceable, stable reference for multi-wavelength interferometry allowing to create multi-wavelength sources with high output power [ 21 , 22 , 23 ]. With this approach recently a comb-referenced four-wavelength-source for distance measurements up to 3.8 m with a combined relative measurement uncertainty of 1.62 × 10 −8 , that was mainly determined by the uncertainty of the empirical compensation of the refractive index was demonstrated and suggested for traceable, precision distance measurements for mass-production of optoelectronic devices [ 23 ]. In this contribution, we introduce the concept of a comb-referenced metrology laser at 633 nm for high-precision homodyne displacement interferometry in a nanopositioning and nanomeasuring machine (NPMM) with a maximum positioning range of 200 mm in a single measurement axis. NPMMs combine high-precision length measurement and positioning of macroscopic objects at nanometer level [ 26 ]. Their development was driven by the need for traceable, dimensional metrology with nm-accuracy for nanotechnology and semiconductor industry at the turn of the millennium [ 26 ]. To this day, different solutions for high-precision full 3D measurement of micro and nano parts based on coordinate metrology have been developed [ 27 ]. At the TU Ilmenau, the NMM-1 with a measurement volume of 25 mm × 25 mm × 5 mm was developed at the turn of the millennium [ 28 ]. Today, this system is commercially available [ 29 ] and it is applied in national metrology and research institutes for the measurement of micro- and nanostructures or international comparisons of step height standards [ 30 , 31 ]. The demand for extended measurement volumina has recently led to the development of the NPMM-200 providing a measurement range of 200 mm × 200 mm × 25 mm with a one-point positioning stability and repeatability of less than 2 nm for lateral measurements over the whole measurement area and a standard deviation of 20 pm for vertical repeatability measurements of a 5 mm step height standard, resulting in a relative resolution of 10 decades considering the full measurement range of 200 mm [ 26 , 32 ]. Currently, one machine is installed at the TU Ilmenau and another one at the Institut für Technische Optik (ITO) in Stuttgart, where it is applied e.g., for the characterization of freeform optics [ 33 ]. The positioning performance of the NPMM-200 sets new demands on the frequency stability of the laser sources used for the underlying interferometric length measurement systems. Currently, polarization stabilized He-Ne gas lasers that still belong to the most widely spread laser sources for precision distance measuring interferometry are deployed [ 34 ]. Their typical relative frequency deviation of 2–6 × 10 −9 over a 24 h measurement results in a measurement error of 0.4–1.2 nm if a maximum measurement range of 200 mm is considered [ 35 ]. Additionally, degradation due to the aging process of the gain medium cause verifiable drifting in the center frequency during lifetime operation of He-Ne lasers [ 36 , 37 ]. Niebauer et al. believe that an effective accuracy of 1 × 10 −9 is technically feasible with two calibrations per year [ 36 ]. Thus, they have to be periodically measured against a Bureau International des Poids et Measures (BIPM)-compliant frequency standard to guarantee full traceability of interferometric length measurements within the NPMM-200 to the SI meter definition [ 35 ]. In this work, we will discuss how a He-Ne laser source permanently tied to a GPS-referenced frequency comb acts as a traceable wavelength standard for displacement interferometry in the NPMM-200 improving the frequency stability of the He-Ne metrology lasers by three orders of magnitude. The approach builds up a permanent link between the interferometric length measurement and an atomic clock and thus the SI second. The paper is organized as follows. The experimental arrangement of the comb-referenced metrology laser will be presented in Section 2 . In Section 2.1 , we describe the initial length measurement system of the NPMM-200 and the traceability chain when using polarization-stabilized He-Ne lasers. Based on this configuration we will derive the necessary properties of a traceable laser source to be integrated into the positioning system of the NPMM. In Section 2.2 , we introduce the metrological fundament for creating ultrastable traceable frequencies at 633 nm. The backbone is a GPS-referenced frequency comb that is accompanied by an Optical Reference System (ORS), consisting of a diode laser locked to a high-finesse cavity. Afterwards we introduce the practical implementation of a comb-referenced He-Ne laser in Section 2.3 and finally describe the methodology for characterizing the frequency stability of the respective laser sources in time domain in Section 2.4 . The main results of our work will be presented and discussed in Section 3 . Therein, Section 3.1 contains a frequency stability analysis of the GPS-referenced frequency comb and in Section 3.2 , we present the results of the comb-stabilized He-Ne source. We discuss the consequences of this stabilization regime on the traceability chain and the impact on length measurement errors within the NPMM-200. In Section 4 , we finally summarize and conclude our work and give an outlook on upcoming experiments and developments. 2. Experimental Configuration and Methods 2.1. State of the Art Length Measuring System of the NPMM-200 The basic metrological concept of the NPMM-200 is presented in Figure 1 and was lately described in [ 32 ]. Further details on the underlying design principles, the drive and guidance system as well as the respective control loops can be found in [ 38 , 39 , 40 , 41 ] and the references given therein. The NPMM-200 applies a high-precision interferometric measuring system to determine the lateral and vertical position of the moving stage and angular deviations of the guidance system [ 32 ]. Figure 1. Setup of the NPMM-200 and its He-Ne laser source for three interferometer axes. The abbreviations in the left picture denote: TMFS—two-mode frequency stabilization, PD—photodiode, PBS—polarizing beam splitter, FC—fiber coupling. The numbers in the right picture correspond to: 1—metrology frame, 2—Positioning stage with corner mirror plate, 3—Sensor mount with nano probe system, 4, 5—Interferometer x , y -axis, 6, 7—Autocollimators (angle sensors). The interferometer of the z-axis is not visible. The base plate below the metrology frame additionally carries the guiding and drive system of the x -, y -, and z -axis (see details in [ 32 , 40 ]). The measuring scale of the displacement interferometers of the x -, y -, and z -axis is determined by the wavelength λ of the corresponding laser which is a function of the frequency ν of the laser and the refractive index of the ambient medium n ambient. Therefore, changes of the laser wavelength are directly considered as length measurement errors Δ L Meas . The uncertainty in the determination of the laser frequency ν and n ambient thus finally limit the precision of length measurements in the NPMM-200. For measurements under ambient conditions, the environmental parameters (pressure, temperature, and humidity) are permanently monitored and the refractive index is calculated from an updated version of the Edlén formula [ 42 ]. Nevertheless, the compensation of the refractive index due to these empirical equations is usually limited to an uncertainty of 10 −7 –10 −8 , mainly caused by uncertainty contributors resulting from the determination of ambient conditions and the air composition [ 43 , 44 ]. Therefore, the NPMM-200 can be additionally operated under vacuum down to 1 mbar to reduce the influence of the refractive index. The signal processing unit of the interferometers utilizes 16-bit A/D-converters for a demodulation of the interferometer signals [ 40 ]. Their resolving capability was determined according to [ 45 ] (see Table 1 in [ 45 ]) with a value of 5.72 pm, assuming an analog-to digital quantization of 16 bit and a rounding error of 16 bit for the arctan function. The interferometers are fiber-coupled and deploy frequency-stabilized He-Ne lasers to minimize the influence of frequency fluctuations on the length measurement. Each of the lasers is independently stabilized by a two-mode comparison technique [ 46 ]. Frequency changes can be achieved by varying the resonator length due to increasing the current through the silver heating coil around the He-Ne tubes (see Figure 1 ). Currently, each of the interferometer axes is powered by one He-Ne laser. These lasers are embedded in a compact, enclosed laser module containing the laser control and stabilization electronics. The He-Ne lasers provide a maximum output power of 700–900 µW per interferometer axis after fiber coupling. The laser module is placed outside the NPMM-200 together with the rest of the NPMM-200 control electronics to avoid heat dissipation from the laser sources (maximum electrical power consumption 60 W) into the NPMM-200 [ 40 ]. Considering a typical measurement interval of 24 h, the polarization-stabilized He-Ne lasers used within the NPMM-200 showed maximum frequency deviations of 1.2–2.7 MHz (relative frequency deviation Δ f / f = 2.5 × 10 −9 –5.7 × 10 −9 ) from the mean value resulting in a measurement error of 0.5 nm–1.1 nm for a maximum measurement range of 200 mm [ 35 ]. Those results are in accordance with other long-term measurements of internally stabilized He-Ne lasers [ 47 ]. Additionally, changes of the center frequency and the shape of the gain profile directly influence the absolute frequency value of the polarization stabilized He-Ne lasers since the control signals for frequency stabilization are directly derived from the gain profile. Therefore, their lifetime wavelength stability is usually limited to 2 × 10 −8 [ 35 , 47 ]. For a measurement range of 200 mm, the corresponding frequency changes of 5–10 MHz would result in a systematic length measurement error of 2–4 nm [ 35 ]. To overcome these errors during long-term operation of the NPMM and guarantee full traceability of the length measurement to the SI meter definition, the absolute frequency value of the metrology lasers has to be periodically measured against a BIPM-compliant frequency standard. For the purpose of frequency calibration, the iodine-stabilized He-Ne laser operating at a wavelength of 633 nm with an uncertainty of u = 10 kHz (2.1 × 10 −11 ) has been mainly used in the visible spectrum for the practical realization of the SI meter definition [ 48 , 49 ]. However, this level of uncertainty can normally only be stated by national metrology institutes (NMI). In Figure 2 , a possible configuration for laser vacuum wavelength calibration including iodine-stabilized He-Ne lasers is presented. To demonstrate the hierarchy between the primary realization of time and the realization of the meter definition by using frequency measurements all possible steps with their respective reported uncertainties are included. National metrology institutes (NMIs) realize the unit of time with the highest accuracy. For the best of these primary frequency standards, relative standard uncertainties in the order of 10 −16 have been reported [ 50 , 51 ]. The NMIs often hold secondary representations of the second such as optical strontium clocks or rubidium oscillators whose frequencies have to be measured against the primary standards leading to relative uncertainties between 10 −14 and 10 −16 [ 50 ]. Other commercially available frequency standards as cesium (Cs) clocks or hydrogen masers are often redundantly operated from the NMIs for time-service applications [ 50 ]. They can furthermore be applied as rf-references for optical frequency combs, determining their uncertainty [ 52 ] in case of, e.g., cesium clocks to a level of 5 × 10 −13 [ 50 ]. Figure 2. Scheme of a possible traceability chain for laser wavelength calibration deploying iodine-stabilized He-Ne lasers. The given uncertainties were taken from the literature as given within the text. Although optical frequency combs are increasingly applied for laser vacuum wavelength calibration, the iodine-stabilized He-Ne lasers are still used for frequency calibration of commercial polarization-stabilized He-Ne lasers and for additional performance tests of GPS-referenced frequency combs (see e.g., [ 53 ]). Nevertheless, their uncertainty of 2.1 × 10 −11 can increase to 8.4 × 10 −11 for systems operating as a secondary standard in industry or research labs [ 54 ]. When measuring a polarization-stabilized against an iodine-stabilized He-Ne laser the uncertainty of the frequency calibration is given by the frequency stability of the specific laser under test [ 47 ]. For polarization-stabilized lasers, relative standard uncertainties (Typ B, k = 2) between 1.3 × 10 −9 and 5.7 × 10 −9 for a 24 h-measurement interval have been reported [ 47 ]. Based on this uncertainty hierarchy of frequency calibration and the described state-of-the-art configuration of the length measurement system, the following requirements for an ultrastable, traceable next generation metrology laser for displacement interferometry in the NPMM-200 can be deduced: For an easy integration into the current length measurement system of the NPMM-200, the next generation metrology laser should be in the VIS near 632.8 nm and provide enough output power (>500 μW) to be suitable for fiber coupling. To practically eliminate the influence of frequency distortions on the length measurement in NPMMs with extended measurement areas of several hundred millimeters, it is desirable that the short-term related length measurement errors fall below the nominal resolution limit of the homodyne interferometers. For the NPMM-200, this limit can currently be set to 2.86 × 10 −11 (5.72 pm at 200-mm measurement range). Taking into account a resolution enhancement in A/D conversion and a possible further extension of measurement ranges up to 1 m, there will prospectively be even a frequency stability of 10 −12 desirable. Ideally, this frequency stability should be available irrespective of integration time. Finally, to control the absolute frequency of the laser source and thus completely trace back the interferometric length measurements, the metrology laser should ideally provide a permanent link to a primary frequency standard [ 55 ]. These requirements cannot be fulfilled with the currently used polarization-stabilized He-Ne lasers. In the following sections, we will thus introduce the new comb-based concept for interferometric distance measurements within the NPMM-200. 2.2. Metrological Basis for Generation of Ultrastable, Traceable Optical Frequencies at 633 nm The commercial availability of traceable, ultrastable optical frequency sources, that have for a long time only been accessible to national metrology institutes, paved the way to create a new metrological base for ultraprecise frequency measurements at the Institute of Process Measurement and Sensor Technology (IPMS) at the TU Ilmenau. The key is a GPS-referenced frequency comb providing exceptional long-term frequency stability and direct traceability to the SI unit second. This fundamental wavelength standard is complemented by an optical reference system (ORS) that is based on a diode laser referenced to an ultrastable high-finesse cavity. Such a laser system provides an exceptional short-term stability and can be used as stand-alone system or as an optical reference for the frequency comb [ 56 ]. In this section, we describe the configuration of both laser systems. 2.2.1. GPS-Referenced Frequency Comb (OFC) The GPS-referenced frequency comb at the IPMS is a commercial system from Menlo System (model: FS 1500-250-WG, Figure 3 ). The comb parameters are phase locked to the frequency output of a GPS-disciplined oscillator (GPSDO). The GPSDO uses the time signal from the GPS (or other GNSS) satellites to steer the frequency output of a local oscillator [ 13 ]. The local oscillator of the GPSDO provides the short-term stability (in this case τ ≤ 100 s) whereas the long-term stability and accuracy of the local oscillator is determined by the GPS signals that are directly traceable to the Universal Time Coordinated ( The United States Naval Observatory ) UTC ( USNO ) [ 13 ]. To transfer the accuracy and stability of the signals from the satellites to the output signal from the local oscillator, the local oscillator is controlled with a servo loop that compares and adjusts the phase of the reference signal from the GPS satellites to the phase of the signal from a voltage controlled ocsillator [ 13 ]. This way the local oscillator is permanently tied to an atomic clock and the GPSDO thus constitutes a “self-calibrating” frequency standard and a cost-effective alternative to an atomic clock. The frequency comb at the TU Ilmenau utilizes an oven-controlled crystal oscillator (OCXO) with a voltage-controlled frequency output of 10 MHz as GPSDO [ 57 , 58 ]. This output is fed to a signal distribution unit (SDU, manufacturer: Meinberg, 12 TTL output ports with 10 MHz, [ 59 ]) that disseminates the reference frequency to the control electronics and data acquisition system of the comb. A schematic of the OFC, the respective control electronics and data acquisition system is depicted in Figure 3 . Figure 3. Structure of the GPS-referenced frequency comb at the IPMS. The abbreviations denote: OFC—optical frequency comb, GPSDO—GPS disciplined oscillator, SDU—signal distribution unit, PD—photodiode. The numbers 1–4 correspond to the respective frequency counter channels. The frequency comb itself is based on a mode-locked, polarization-maintaining erbium fiber laser with a center wavelength of 1552 nm and a default repetition rate of f Rep = 250 MHz that can be adjusted between ± 1 MHz [ 60 ]. A 2 f - f interferometer configuration is used for the detection of the CEO that is set to f CEO = 20 MHz by default [ 60 , 61 ]. For the 2 f - f interferometer, one part of the laser output is amplified in an erbium-doped fiber amplifier (EDFA) and afterwards spectrally broadened in a highly nonlinear fiber (HNLF). The frequency comb provides six output ports at 1550 nm with a maximum power output of 18 mW and a spectral width (FWHM) of 20 nm. Additionally, the comb is equipped with a second amplifier unit that generates a 633 nm output by spectral broadening in a HNLF and subsequent frequency doubling. This output provides an average power of 7 mW and a spectral width of 3 nm (FWHM). It can be used to create a beat signal with laser sources at 633 nm, e.g., He-Ne lasers. The data acquisition system of the comb is equipped with four Π-type frequency counters having a gate time of τ = 1 s (model: FXM50) [ 62 ] and two spectrum analyzers (model: HMS-X, Rohde & Schwarz). The frequency counters are referenced by the 10 MHz output of the SDU. The comb parameters f Rep and f CEO are monitored with two frequency counters. The remaining frequency counters can be used for the counting of beat frequencies with external laser sources. The feedback loops of the comb phase lock the repetition rate and the CEO to a multiple of the 10 MHz-frequency output [ 60 ]. A detailed description of the control loops can be found in [ 35 , 60 ]. An undisturbed reception of the GPS signal together with a permanent closed loop operation of the local oscillator are the basic prerequisite for a sufficient stabilization of the comb parameters and thus the generation of stable, traceable optical frequencies [ 14 , 35 ]. To control the locking status of the comb parameters, f Rep and f CEO are permanently measured by the frequency counters of the data acquisition system after the respective servo loops have been closed. For a continuous monitoring of the incoming GPS signals we extract data about the number of available satellites and the overall status of the GPS signals from the transmitted data files via the serial interface of the GPSDO. During a typical 24-h operation, we found that the number of available satellites usually varies between 6 and 10 which is sufficient for a distortion-free operation of the GPSDO. When proper operation of the GPDSO is assured, the manufacturer guarantees a relative accuracy better than 8 × 10 −12 (τ = 1 s) and a relative frequency stability (relative Allan deviation) A Dev better than 4 × 10 −12 [ 57 ]. To determine the absolute frequency of external laser sources, the specific laser under test is superimposed with a comb line and the time dependence of the generated heterodyne signal is analyzed (see Section 2.4 ). For this purpose, the OFC is equipped with a free-space beat detection unit (FS-BDU) consisting of polarizing beam splitters, half-wave-plates, a diffraction grating and an avalanche photodiode (Model: APD210, Menlo Systems) for spatially overlapping the beams from the frequency comb and the external laser source (see e.g., [ 35 ]). For the detection of the heterodyne time traces, one of the counters of the data acquisition system is used. 2.2.2. Optical Reference System (ORS) The optical reference system is an ultrastable laser system based on a CW laser stabilized to a high-finesse cavity. The cavity serves as a reference with an exceptional short-term frequency stability [ 63 ]. In contrast, their long-term stability is limited by frequency drifts due to aging and temperature fluctuations of the cavity [ 63 , 64 ]. At the IPMS, a commercial system from Menlo Systems is under operation since 2021 [ 65 ]. The basic setup of the ORS is depicted in Figure 4 . The ORS consists of an external cavity diode laser (ECDL, manufacturer: MOGLabs, Littrow configuration; wavelength: 632.8 nm [ 66 ]) that is locked to a cylindrical cavity made of ultra-low expansion (ULE) glass ceramics via the Pound–Drever–Hall (PDH) locking scheme [ 67 , 68 ]. The ULE spacer has a length of 121 mm and is equipped with high-finesse ion beam sputtered ULE mirrors [ 69 ]. The ULE glass exhibits a linear shrinking drift rate of approximately 0.15 Hz/s [ 69 ]. The optical cavity is temperature stabilized and embedded in an ultra-high vacuum (UHV) system with an achievable pressure of 7 × 10 −9 mbar. The optics required to couple the light from the ECDL into the cavity and the opto-electronical components needed for the implementation of the PDH-Lock (electro-optic modulator for generation of sidebands at 20 MHz, quarter-waveplate, half-wave plate, polarizing beam splitter, photodetector, see Figure 4 ) are mounted on a breadboard vertically attached to the vacuum chamber. The cavity and all opto-mechanical parts are placed in an acoustic isolation box, mounted on an additional vibration isolation platform [ 69 ]. A control unit contains all electronics necessary for operating, tuning and locking the laser as well as the electronics for the temperature stabilization of the cavity [ 69 ]. The ECDL, ULE-cavity with optical platform and high vacuum pump and the control electronics are integrated into a compact 19” rack. The system provides an output power of 10 mW that can be accessed via a fiber-coupled, polarization-maintaining output port [ 69 ]. The ORS can be used either as stand-alone system or as an optical reference for the OFC (see e.g., [ 70 ]). Figure 4. Structure of the ORS. The abbreviations denote: ECDL—external cavity diode laser, FC—fiber-coupled, NBS—neutral beam splitter, C—collimator, PMF—polarization-maintaining fiber, M—mirror, EOM—electro-optic modulator, HWP—half wave plate, PBS—polarizing beam splitter, QWP—quarter wave plate, L—lens, DDS—direct digital synthesizer, LPF—low pass filter. The red lines indicate optical path lengths, the dashed gray lines electronic signals. The light of the ECDL is sent through a phase-modulator operating at 20 MHz to generate sidebands for the PDH-lock. The 20-MHz reference signal is provided by a DDS. The error signal is created by mixing the signal from the photodetector containing the reflected light from the cavity with the signal from the DDS and subsequent low-pass filtering. The frequency of the laser is stabilized via fast feedback to the current and slow feedback to the piezo-electric actuator controlling the external cavity length of the diode laser [ 65 ]. In Figure 5 , the performance of the ORS-system as provided by the manufacturer is presented. In Figure 5 a, the frequency deviation of the heterodyne beat signal between two optical reference systems of comparable frequency stability is presented. The data are already corrected for the drift rate of the cavity [ 71 ]. As can be seen from the corresponding relative Allan deviations depicted in Figure 5 b, the ORS provides an excellent short-term stability of A Dev = 3 × 10 −15 at τ = 1 s [ 71 ]. Within the frame of the present work, we will apply the ORS as an ultrastable reference laser to characterize the short-term frequency instability of the OFC. This allows for the first time to directly resolve the instability behavior of our GPS-referenced frequency comb. For this purpose, the output of the ORS is heterodyned with one of the comb modes in a fully fiber-coupled beat detection unit (FF-BDU) [ 69 ]. Figure 5. Frequency stability of the ORS as retrieved from manufacturer data (M.d.). ( a ) Time dependent beat frequency between two optical reference systems of comparable frequency stability with linear drift removed, ( b ) corresponding relative Allan deviation (see Section 2.4 ). 2.3. Comb-Referenced, Fiber-Coupled He-Ne Laser Source For an easy integration of a comb-referenced metrology laser into the current length measurement system of the NPMM-200, we decided to build-up a He-Ne source similar to the laser module presented in Figure 1 . Nevertheless, due to the limited bandwidth of the Ne gain profile, the approach of locking three independent lasers to individual comb lines as applied in comb-referenced multiwavelength interferometry is not a suitable approach for the He-Ne lasers used within the NPMM-200. Instead, we decided to implement a laser-array configuration as originally described in [ 72 ]. The laser module of the NPMM-200 was thus extended with a fiber-coupled digitally frequency offset-locked He-Ne heterodyne source consisting of two commercially available He-Ne lasers (model: SL02/1, SIOS Meßtechnik GmbH [ 73 ]) with the same mechanical design and similar transfer behavior [ 74 ]. The basic control concept of this heterodyne source is indicated in Figure 6 and was described in detail in [ 74 ]. The heterodyne source consists of two sealed internal mirror He-Ne laser tubes that are covered by a protective aluminum chassis and can be independently stabilized by a two-mode comparison technique using [ 74 ]. The respective optics and control electronics are integrated into the aluminum chassis and are provided by the manufacturer [ 73 ]. Both lasers are mounted on a U-profile which holds the beam splitters for beat frequency generation, Faraday isolators to to prevent back reflections into the laser tubes, fiber couplers, and the polarization maintaining optical fibers. Prior to fiber coupling, the lasers provide an output power of approximately 1.2 mW [ 73 ]. To adjust the frequency offset between the two lasers, some µW optical power are branched off with beam splitters to generate a beat signal with high SNR of approximately 50 dB. The beat signal is detected with an avalanche photo diode (Hamamatsu C5658). Due to the high SNR of the beat signal, a conventional edge-detection method can be used for frequency determination [ 74 , 75 ]. For this purpose, the analog signal from the photodiode is transformed into a TTL-signal via a Schmitt–Trigger circuit and is subsequently fed into an FPGA-based control loop (card: PXI-7853R and chassis PXI-1042Q). Therein, the number of counted pulses within a well-defined time frame is compared to a user defined frequency determining the frequency offset between the two lasers. The resulting frequency difference is fed into a digital PID-controller and the digital output signal is converted into an analog voltage [ 74 , 75 ]. The analog voltage is finally transformed into a current by a transconductance amplifier (U/I converter (UIC)) and drives the heater of the second laser tube to adapt its cavity length to follow the frequency of the first laser [ 74 , 75 ]. If the first laser is kept in its two-mode frequency stabilization regime, the maximum frequency deviation over one day limits the frequency stability of the heterodyne source to 2 × 10 −9 (see Section 3.2 ). Figure 6. Layout of the comb-referenced He-Ne heterodyne source. The abbreviations denote: GPSDO—GPS disciplined oscillator, SDU—signal distribution unit, APD—avalanche photodiode, PBS—polarizing beam splitter, M—mirror, BSA—beam splitter assembly, FI—Faraday isolator, FC—fiber coupling, UIC—transconductive amplifier (U/I converter), FPGA—field programmable gate array. To realize a comb-referenced He-Ne source, the first laser of the heterodyne-source has to be permanently locked to a single comb line. This is schematically shown in Figure 6 . Since the optical power per comb line is low (approx. 800 nW per comb mode before passing optical components for beat signal detection), almost the entire output power of the first laser is used for generating a beat note with a respective comb mode [ 35 ]. The resulting beat signal with an SNR of 32–34 dB prevents the application of an edge-detection method. Thus, a phase-sensitive detection is applied for the frequency determination of the beat signal. For this purpose, the beat signal detected with an avalanche photodiode is fed to the analog input port of an FPGA-based control system (controller: NI-7931R and transceiver module: NI-5782). The input signal is converted by an analog-to-digital converter (ADC) of the control system with a sampling rate of up to 250 MS/s and is further processed by the embedded FPGA unit (model: Xilinx Kintex-7-K325T). The control electronics are synchronized via the 10-MHz output of the GPSDO. The implemented servo loop allows to correctly determine the beat frequency even at a low SNR ≤ 10 dB. The detection algorithm is currently designed with a ± 1-MHz bandpass filter around a fixed center frequency of 62.5 MHz [ 75 ]. The phase data of the input signal is used to generate a frequency variable by subtracting subsequent values over a time slot of 64 ns. The resulting frequency value is low-pass-filtered and finally forwarded to a PID controller algorithm [ 75 ]. The resulting digital output signal is afterwards converted into a DC coupled voltage signal using a digital-to-analog-converter (DAC) with a resolving capability of 20 bit (model: AD5791, Analog Devices) [ 75 ]. Finally, the voltage signal is converted into a current by a second transconductive amplifier. This current drives the heating coil of the first laser of the heterodyne source to change its resonator length and thus adapting its frequency to follow the frequency of the respective comb line [ 75 ]. This way, the He-Ne laser directly stabilized to a comb line serves as a “Secondary Standard Laser” (SSL) as depicted in Figure 6 and the second laser of the heterodyne-source tied to the SSL serves as the actual metrology laser (ML). The specific locking procedure and determination of the absolute frequency of both laser sources is described in detail in [ 35 ]. The remaining optical power of 500–700 µW from the metrology laser is finally used to feed the two 200-mm interferometer axes (x and y) of the NPMM-200. In opposition to the original configuration shown in Figure 1 , this allows to transfer the same laser frequency to the x - and y -interferometer axes of the NPMM-200. To disseminate the comb-stabilized frequency output to the interferometers of the NPMM-200 a polarization maintaining (PM) 50:50 fiber coupler and a 6 m polarization-maintaining fiber is used. 2.4. Analysis of Frequency Instability For the analysis of the frequency instability of the GPS-referenced frequency comb as well as the OFC-locked He-Ne lasers, we use the heterodyne method. For this purpose, the GPS-referenced frequency comb is superimposed with the ORS or the He-Ne lasers within the FF-BDU or the FS-BDU as described within the prior sections. Subsequently, the respective beat signals of the APD are low-pass filtered, amplified, and band pass filtered (3-dB bandwidth 49.8 MHz… 70.5 MHz around the center frequency of 60 MHz, [ 76 ]). The bandpass-filtered signal is fed to the frequency counters and the recorded frequency-over-time-traces are used to analyze frequency instability in the time domain. To study frequency instability in time-domain, the Allan variance σ y 2 τ is calculated for a discrete, finite series of N frequency values f i averaged over a specific sampling time τ that can be obtained as an integer multiple m of a basic measurement interval τ 0 [ 77 , 78 ]: σ y 2 τ = 1 2 N − 1 ∑ i = 1 N − 1 f i + 1 − f i 2 . (2) The basic measurement interval is determined by the gate time of the FXM50 frequency counter used throughout this work and corresponds to τ 0 = 1 s. For practical calculations of the Allan variance, usually overlapping samples are used to improve the confidence of the stability estimate and the results are expressed as the square root of σ y 2 τ , the Allan deviation A Dev [ 78 , 79 ]. 3. Results and Discussion 3.1. Frequency Instability of the GPS-Referenced Frequency Comb The time traces of the frequency measurements of the comb parameters and the heterodyne signal between the respective comb line and the ORS are shown in Figure 7 for the free-running comb over a 4-h measurement interval and in Figure 8 and Figure 9 for an in-loop operation of the comb. For better readability, the changes in repetition rate in Figure 7 a are depicted as deviation from its initial value. The free-running repetition frequency shows a slight drift of 22 Hz within the observed time window. Although the respective changes in the CEO within the same observation time are within the MHz-range, the resulting frequency change of the respective comb line is completely determined by the change in repetition rate as can be deduced from the frequency drift of the beat frequency depicted in Figure 7 b. Due to the large multiplication of the repetition frequency into the optical domain as described by Equation (1), the rather small frequency drift of the repetition rate results in a 42 MHz (Δ f / f = 8.9 × 10 −8 with f HeNe = 473.6127 THz) frequency change of the respective comb mode ( n = 1,894,861) that is reflected in the frequency drift of the beat frequency between the comb line and the ORS. The opposite sign of the slope is caused by the sign of the beat signal. Since the ORS frequency lies above the considered comb line, an increase in the repetition rate leads to a decrease of the beat frequency as demonstrated in the inset of Figure 7 b. Figure 7. Frequency instability of the free-running comb. ( a ) Time traces of the free-running comb parameters measured with the FXM50. The left y -axis depicts the change of repetition rate from its initial value. The right y -axis the CEO. ( b ) Time trace of the beat note between the respective comb line and the ORS. Since the frequency υ ORS of the ORS lies above the respective frequency of the comb line υ n , an increase in the repetition rate f Rep results in a decrease of the beat frequency f Beat . To monitor the frequency changes of the beat note, the measurements were performed without the bandpass filter at 60 MHz. Figure 8. In-loop comb parameters of the GPS-referenced frequency comb. ( a ) Time trace of the locked repetition rate. ( b ) Time trace of the locked CEO. Both plots depict the frequency deviations from the mean value over a measurement time of 24 h. ( c ) Calculated Allan deviations. The gray and blue lines present a linear fit to the data as described within the text. Figure 9. Frequency instability of an in-loop GPS-referenced frequency comb. ( a ) Time trace of the heterodyne signal between the OFC and the ORS. The red line indicates a linear fit to the data with f = 62.384 MHz–0.067 Hz/s. ( b ) A 1 h section of the frequency fluctuations of the heterodyne signal after the linear drift was removed from the measured data. ( c ) The respective relative Allan deviation of the heterodyne signal obtained by dividing the Allan deviation by f HeNe ≈ f Comb ≈ 474 THz. The abbreviation M.d. denotes “manufacturer data”. In Figure 8 a,b, the time traces of f Rep and f CEO locked to the rf reference are presented. Both comb parameters follow the reference frequency provided by the GPSDO with a maximum frequency deviation from the mean value of 2 mHz (Δ f / f Rep = 8 × 10 −12 ) for f Rep and 6 Hz (Δ f / f CEO = 3 × 10 −7 ) for f CEO . The respective Allan deviations in Figure 8 c present the tracking stability of the comb parameters with a τ −1 -dependence for both parameters. This corresponds to white phase noise characteristics as supported by the modified Allan deviation showing a τ - −3/2 -dependence (see e.g., [ 78 ]) for integration times below 10,000 s (not shown Figure 8 c). To analyze the resulting frequency instability of the respective comb line, the time trace of the heterodyne signal between the OFC and the ORS and the respective relative Allan deviation are presented in Figure 9 . In Figure 9 a, a clear frequency drift is evident when measuring the beat frequency over a period of 5 days. This frequency drift is mainly caused by the frequency drift of the cavity material of the ORS. To a first approximation it can be described by a linear fit with a slope of 67 mHz/s, resulting in a frequency change of 5.8 kHz (Δ f / f = 1.2 × 10 −11 , f = 474 THz) in 24 h and an overall frequency drift of 28.9 kHz within the observation time of 5 days. The frequency drift of the ORS cavity thus dominates the long-term stability of the heterodyne signal as can be deduced from the increase of the Allan deviation for τ > 4000 s in Figure 9 c and the data points of the Allan deviation of a linear drift rate introduced as a red line in Figure 9 c. Nevertheless, the observed drift rate is much smaller than the drift rate of 150 mHz/s as reported by the manufacturer [ 69 ]. This can be attributed to aging effects of the cavity material as reported in [ 64 ] and is consistent with prior measurements where a drift rate of 104 mHz/s was obtained [ 80 ]. Comparing the Allan deviation of the beat signal with the data for the ORS as given by the manufacturer thus clearly reveals that the Allan deviation for τ < 4000 s reflects the frequency instability of the GPS-referenced frequency comb. Therefore, the heterodyne signal mainly represents frequency fluctuations of the respective comb line when the drift rate is removed as indicated in Figure 9 b. Within the measurement interval of 5 days, a maximum frequency deviation of 25.2 kHz (Δ f / f = 5.3 × 10 −11 ) was observed from the drift-corrected frequency values of the beat signal. Over a timeframe of 1 h, a typical maximum frequency deviation of 14.8 kHz (Δ f / f = 3.1 × 10 −11 , obtained as a mean value of 1-h sections within the 5-day time frame for τ = 1 s) occurred. The measurement data of the unlocked comb presented in Figure 7 demonstrated the influence of small frequency changes of the repetition rate on the overall frequency instability of a comb line. Since the measurement of the comb parameters revealed a high tracking stability ( Figure 8 ), the Allan deviation of the heterodyne signal between the comb line and the ORS thus essentially reflects the frequency instability of the GPSDO. For comparison, Figure 9 additionally contains Allan deviation data provided by the manufacturer (gray line in Figure 9 c). This dataset was obtained from a 13-h measurement series of a comb–comb comparison with two GPS-referenced frequency combs [ 57 ]. For short integration times, the Allan deviation increases with a slope of τ 0.2 , reaching a distinct maximum of 3.6 × 10 −12 at τ = 64 s. For longer integration times, the Allan deviation decreases. This behavior is consistent with former results reported in the literature [ 81 , 82 , 83 , 84 ]. The frequency output of the OCXO is steered to agree with the signals transmitted by the GPS satellites and can thus take advantage of their high long-term stability [ 13 , 84 ]. The Allan deviation obtained from the frequency measurement between a comb line and the ORS starts to coincide with the manufacturer data at τ = 64 s. For smaller integration times, considerably higher Allan deviations are obtained from the ORS-OFC comparison. In opposite to the manufacturer data a maximum Allan deviation of 4.6 × 10 −12 at τ = 8 s is observed. The origin of this discrepancy is currently not clear. We did not find any indications that the settings of the comb parameter control loops (e.g., a too high P-gain) or the additional integration of the SDU led to these differences. Therefore, further investigations are needed to clarify the origin of this degradation in short-term-frequency stability compared to the manufacturer data. Since the accuracy of the GPSDO over a given period of time is usually limited by its stability, it is common sense to use twice the Allan deviation for a time duration that orientates towards the duration of frequency calibration to determine the frequency uncertainty of a GPSDO [ 82 ]. Covering a worst-case scenario, manufacturer data rather tend to “overestimate” uncertainty by relying on the highest Allan deviation reported (see Section 2.2 ). Taking these considerations into account and regarding that the frequency instability of a comb line is primarily determined by the frequency instability of the used rf reference (see e.g., [ 52 ]), we have to claim an uncertainty of 9.2 × 10 −12 (k = 2, τ = 8 s) for the GPS-referenced frequency comb at the TU Ilmenau. Therefore, reaching an uncertainty below 10 −12 makes an integration time of at least 8000 s necessary. 3.2. Frequency Instability of the Comb-Referenced He-Ne Source In Figure 10 and Figure 11 , the frequency stability of the comb-referenced He-Ne sources is finally presented. Figure 10 presents a measurement series of the He-Ne heterodyne source when both lasers are in their internal two-mode comparison stabilization regime and are measured against an optical comb line. In this case, the comb serves as a stable reference laser revealing the frequency instability characteristics of the He-Ne heterodyne source. In Figure 10 , the frequency fluctuations over a typical measurement interval of 24 h obey a long-periodic frequency change that is superimposed by several frequency jumps up to 1.5 MHz. The long-term frequency changes lead to a rise of the Allan deviation for τ > 100 s resulting in a deviation of 3–4 × 10 −10 at τ = 8000 s. The first laser serving as a secondary standard laser (SSL) obeys a maximum frequency deviation of 1 MHz. Although both lasers are identical in construction and their long-term frequency instability is comparable as deduced from the intersection of the Allan deviation for τ > 100 s, they obey a different frequency stability behavior within the short-term regime as can be seen from the frequency fluctuations in Figure 10 a,b as well as the Allan deviation below 100 s. Nevertheless, the frequency instability of both lasers is consistent with the results we recently reported for the lasers of the He-Ne module of the NPMM-200 [ 35 ]. Thus, the heterodyne source can be considered as a suitable representative of the frequency characteristics of the NPMM-200 laser module and an appropriate candidate for the realization of a comb-referenced laser module. Figure 10. Frequency instability of the He-Ne heterodyne source used as a starting point for the realization of a comb-referenced laser module. ( a ) A 24 h-measurement series of the secondary standard laser (SSL) against a comb line, ( b ) 24 h measurement of the metrology laser (ML). In each of the plots, the frequency deviations from the mean value derived over the whole measurement series of 24 h are shown. ( c ) Relative Allan deviations obtained by dividing the frequency fluctuations by a nominal value of 474 THz. Figure 11. Frequency instability of the comb-referenced-He-Ne heterodyne source. ( a ) 24-h measurement series of the secondary standard laser (SSL) prior and after closing the control loop (closed-loop operation reproduced from [ 35 ]). ( b ) Relative Allan deviations. The Allan deviation of the OFC is obtained by combining the Allan deviation as calculated from the measurement of the beat signal between the ORS and the OFC for integration times below 4000 s and the Allan deviation given by the manufacturer for integration times above 4000 s. ( c ) The 1 h measurement sections of the 24-h timetrace. The upper graph depicts the SSL locked to the comb and the frequency fluctuations of the drift-removed beat signal between a comb line and the ORS (taken from Figure 9 b) for comparison. Please note that in contrast to the in-loop measurements of the heterodyne source, those measurements were not taken simultaneously. The graph in the middle enlarges the frequency scale to illustrate the frequency fluctuations of the SSL when locked to the comb line. The lower graph further enlarges the frequency scale to depict the fluctuations of the beat signal between the ML and the SSL, when locked to the comb line. All three graphs show the same 1 h section and present the frequency deviations from the respective mean value of the whole measurement series. The final closed loop operation of the comb-referenced He-Ne source is depicted in Figure 11 . In Figure 11 a, the deviation of the beat frequency between the SSL and a comb line from its mean value Δ f Beat = f Beat − f B e a t ¯ after closing the control loop is shown. Additionally, the changes of the heterodyne signal when the SSL is in its internal stabilization regime are plotted for comparison from Figure 10 a. When the control loop between the comb line and the SSL is closed, the frequency deviations of the SSL are eliminated and the beat signal reflects the tracking stability of the SSL in relation to the comb line [ 75 ]. In Figure 11 c, a 1 h-section of the measurement series is enlarged. The upper graph of Figure 11 c additionally contains the changes of the beat signal of the comb line obtained from the measurement data of the beat frequency between a comb line and the ORS as discussed in Section 3.1 . Please note that these two measurements were not taken simultaneously and their absolute frequencies correspond to two different comb lines due to the different absolute frequencies of the ORS and the He-Ne lasers. In the central and lower graph of Figure 11 c, this 1-h section is further enlarged to make the frequency deviations of the beat signals between the SSL and the comb line as well as the ML and the SSL visible when both control loops were closed. Therein, the bright green line represents the frequency deviation from the mean value of the beat signal between the SSL and the ML and thus demonstrates how the ML is following the SSL when the SSL is locked to the comb line. Considering the whole 24-h measurement series occasional outliers resulting in a maximum frequency deviation of 11.7 kHz were observed [ 35 ]. These outliers stem from severe acoustic noise due to slamming of doors and are to be avoided within a normal operation of the comb-referenced He-Ne lasers and the NPMM-200. The 1-h section in Figure 11 c presents a part of a 10-h measurement window where no extreme external disturbances occurred. Based on this dataset, we observed on average a maximum frequency deviation of 1.8 kHz (Δ f/f = 3.8 × 10 −12 with f = 474 THz) with a standard deviation of 146 Hz for the in-loop heterodyne signal between the SSL and the comb line and of 62 Hz (Δ f / f = 1.3 × 10 −13 ) with a standard deviation of 5 Hz for the in-loop heterodyne signal between the SSL and the ML within a 1 h measurement window (values are obtained as a mean value of 1-h sections within the 10-h measurement window for τ = 1 s). On the other hand, the deviations of the mean value of the measured beat frequencies from the nominal values of the closed-loop control are −47 Hz (Δ f / f = 9.9 × 10 −14 ) at f 0 = 62.5 MHz for the SSL and 204 Hz (Δ f / f = 4.3 × 10 −13 ) for the ML at f 0 = 4.5 MHz [ 35 ]. Comparing this to the observed maximum frequency deviations from the mean value, it can be deduced that the accuracy of the ML control loop is limited by a frequency offset and not the frequency stability of the beat signal. The corresponding relative Allan deviations of the measurement series are presented in Figure 11 b. For a better comparison of the frequency instability of the in-loop beat signals to the Allan deviation of the comb line, all values were related to a nominal frequency of 474 THz. The black curve depicts a combination of the Allan deviation of the frequency comb as obtained from the measurement of the beat signal between the ORS and the OFC for integration times below 4000 s and the manufacturer data for integration times above 4000 s. The relative Allan deviation of the beat signals between the SSL-OFC and the ML-SSL both fall below the Allan deviation of the comb line for integration times τ ≥ 1 s. This again demonstrates that both servo loops enable the He-Ne lasers of the heterodyne source to follow the comb line. The Allan deviation of the beat frequency between the SSL/comb line (SSL/ML) fall with a slope of τ −0.8 ( τ −0.6 ) for integration times between 2 s and 500 s that flattens down to a τ −0.2 –dependence for integration times above 1000 s. The frequency stability of the comb-referenced metrology laser is thus finally determined by the Allan deviation of the comb line. The frequency fluctuations and differences in the Allan deviation of the beat signals between the SSL/comb line and the SSL/ML furthermore indicate that the limited and similar dynamic behavior of the laser control systems produces some lowpass filtering effects on the higher frequency distortions of the comb line. In this case, the locked SSL would not be able to follow fast comb line jitters leading to a “smoothing” out of frequency distortions of the comb line [ 75 ]. To prove this thesis, we now prepare direct measurements of the He-Ne-locked system against the ORS and concentrate on more detailed investigations on the frequency noise properties of the comb line and the in-loop-comb-referenced metrology laser. Finally, comparing the Allan deviation of the comb line with the Allan deviation of the SSL that represents the previous status of frequency stability in the NPMM-200, an improvement of nearly two orders of magnitude at τ = 1 s and of up to three orders of magnitude at τ = 10,000 s can be achieved. Thus, the approach of a comb-referenced He-Ne laser source allows a significant improvement of the long-term frequency stability of these lasers and additional changes in absolute frequency due to aging effects can permanently be monitored and avoided once their absolute frequency has been determined as described in [ 35 ]. As a result, the traceability chain for the measurement of laser frequency is significantly changed in comparison to Figure 2 . The NMIs contribute to the calculation of the UTC by sending data from their local clocks to the BIPM [ 82 ]. The GPS satellites carry atomic oscillators and are controlled by the United States Department of Defense (U.S. DoD) [ 13 ]. The GPS time is referenced to the UTC time scale maintained at the United States Naval Observatory UTC ( USNO ) which is the largest contributor to the calculation of the UTC [ 82 ]. This way, the GPS receivers generate output signals that agree with UTC ( USNO ) [ 82 ]. Within the GPSDO, the output signals from the GPS receiver are used to discipline the local oscillator providing a frequency output with an uncertainty that is mainly determined by the frequency instability of the locked oscillator [ 13 , 82 ]. The comb technology allows to transfer the accuracy and stability of the GPSDO into the optical domain, where it is finally delivered to the length measuring system of the NPMM-200 by the comb-referenced He-Ne source. Thus, a direct and permanent link to the SI unit second can locally be established as schematically shown in Figure 12 . Figure 12. Scheme of the reduced traceability chain for laser wavelength calibration of the metrology lasers of the NPMM-200. Nevertheless, it has to be mentioned that the current locking scheme does not compensate any potential frequency fluctuations due to environmental disturbances within the fiber link transporting the frequency of the comb-referenced metrology laser to the length measurement system of the NPMM-200 as discussed in Section 2.3 . Since these disturbances are mainly caused by thermal noise and their impact increases with fiber length, we chose the fiber length as short as possible (see e.g., [ 85 ] for fiber thermal noise). The comb and the heterodyne source are placed in the same air-conditioned lab as the NPMM-200 keeping the impact of temperature variations as small as possible. Based on a worst-case-scenario calculation retrieved from [ 86 ], where a maximum temperature change of 7 mK/s obtained from temperature data within the lab was assumed, a maximum frequency change of 8 × 10 −16 per meter fiber length was retrieved [ 35 ]. Compared to the Allan deviation of our GPS-referenced frequency comb, this frequency change is neglectable for the 6-m fiber length for integration times below 10,000 s. Nevertheless, if prospectively longer fiber paths covering several ten meters of fiber length for a further internal distribution of the ultrastable frequencies are considered, the validity of our assumption has to be confirmed within further experiments on the phase-noise characteristics of the comb-referenced fiber-coupled He-Ne laser source. From the current point of view, the overall uncertainty of the comb-referenced metrology laser is thus determined by the frequency instability of the respective comb line for integration times up to 10,000 s. In Figure 13 , finally the influence of frequency instability on the length measurement within the NPMM-200 is discussed. In Figure 13 a,b, the expected frequency fluctuations of the comb line obtained from the heterodyne signal between the OFC and the ORS are transferred into a length measurement error using the interferometer equation for a Michelson interferometer configuration [ 87 ]: L = N · c 2 · e · n · f (3) where L describes the measured displacement, N the respective counter value that corresponds to a change in interference order, c the velocity of light, e an electronic interpolation factor, n the refractive index of the ambient medium, and f the frequency of the laser source. By applying mathematical differentiation on Equation (3) with respect to frequency and subsequent dividing by L to obtain a relative expression on the resulting length measurement error one obtains: Δ L L = − Δ f f (4) Figure 13. Influence of frequency distortions on the interferometric length measurement within the NPMM-200. ( a , b ) transform the frequency fluctuations and uncertainty of the comb line into a theoretical length measurement error for a maximum measurement range of L = 200 mm. ( c , d ) experimentally demonstrate the influence of frequency changes on the interferometric length measurement within the NPMM-200 at a constant distance of L = 100 mm using the comb-referenced He-Ne laser as a tunable laser source, ( c ) equidistant frequency steps of Δ f = 1.25 MHz and ( d ) repeated frequency jumps of Δ f = 0.625 MHz height. Integration time 0.5 s [ 35 ]. In Figure 13 , Equation (4) is used to calculate Δ L for a maximum measurement range of L = 200 mm where for simplicity the absolute value of Δ f / f is used. As can be seen in Figure 13 a, even regarding a worst case scenario where a complete transfer of frequency fluctuations of the comb line to the He-Ne laser is assumed, the fluctuations of the comb line mainly stay below the nominal resolution of the current signal processing unit of the NPMM-200. In Figure 13 b, the expected length measurement error was calculated based on the uncertainty of the comb as discussed in Section 3.1 . Therein, a maximum length measurement error of only 2 pm is expected for L = 200 mm demonstrating that the influence of frequency distortions fall below the nominal resolution of the current interferometers within the NPMM-200 for integration times above 1 s and can thus be neglected. This is also true if the additional tracking instability of both beat signals would be added to the relative Allan deviation values underlying this calculation. For comparison, Figure 13 b additionally plots the length measurement error obtained from twice the Allan deviation of the He-Ne laser when stabilized by the two-mode comparison technique. This presents the former status within the NPMM-200 and demonstrates the not negligible influence of the long-term stability on the length measurement. Nevertheless, the negligible length measurement errors expected from the comb-referenced He-Ne lasers makes a direct experimental study on their impact within the NPMM-200 difficult. Therefore, as an indirect proof of our approach, we used the tunability of the comb-referenced He-Ne source to directly demonstrate and separate the influence of frequency distortions on the interferometric length measurements. For this purpose, we intentionally introduced frequency changes by varying the beat frequency between the ML and SSL when both lasers are locked to the comb line. For this measurement, the positioning stage of the NPMM-200 was mechanically fixed at a distance of L = 100 mm and the drive system of the NPMM-200 was deactivated. The measurements were taken under normal atmospheric conditions without evacuating the NPMM-200 chamber. During the measurements either equidistant frequency steps or frequency jumps where applied. The absolute value of the frequency changes was adapted to correspond to the typical frequency changes we observed when the He-Ne lasers are in their internal stabilization mode based on the two-mode comparison technique as discussed in Figure 10 . The results of these measurements are presented in Figure 13 c,d. According to Equation (4), the subsequent frequency steps of 1.25 MHz in Figure 13 c should lead to a frequency-related length change of 264 pm and in Figure 13 d repeated, individual frequency jumps of 625 kHz should cause a length change of 132 pm for the considered measurement range of 100 mm. Experimentally, we observed a mean length change of 0.25 nm with a standard deviation of 0.03 nm per frequency step for the frequency staircase, resulting in an overall frequency change of 6.25 MHz that corresponds to a frequency related length change of 1.3 nm as clearly visible in Figure 13 c [ 35 ]. The deviations from an ideal staircase observed in Figure 13 c indicate that disturbances are not only caused by the introduced frequency changes but also contain mechanical vibrations, temperature fluctuations, and related changes in the refractive index making a baseline correction of the data necessary. In case of the up-and-down frequency jumps, an average length change of 133.5 pm with a standard deviation of 9.9 pm was retrieved from the measured data after averaging down the measured length data with an integration time of 0.5 s [ 35 ]. 4. Summary and Outlook Ultrastable and traceable wavelength standards are a key component for high-precision displacement interferometry in nanopositioning and nanomeasuring machines. With the demand for extended measurement volumina, the respective wavelength standards have to meet increasingly higher demands as well. Based on the state-of-the-art length measuring system of the NPMM-200 with a measurement volume of 200 mm × 200 mm × 25 mm, we deduced the requirements for such a wavelength standard and described how it was experimentally implemented by means of a comb-referenced He-Ne heterodyne source at 633 nm. The key component, a GPS-referenced frequency comb, serves as a “self-calibrating” frequency standard providing direct traceability to the SI unit second and an exceptional long-term frequency stability of 5 × 10 −13 at τ = 10,000 s. By locking the He-Ne lasers to a comb line, it was possible to improve their long-term frequency stability by three orders of magnitude. This approach allows us to establish a traceable and permanent link of interferometric length measurements to the SI unit second. With the improved frequency stability of the comb-referenced gas lasers, the influence of frequency-related length measurements errors practically drops below the nominal resolution of the signal processing unit of the NPMM-200 for the first time. This allows to neglect the laser frequency as an uncertainty contribution factor within our length measurements. However, our experiments also revealed limitations of the implemented GPS-referenced wavelength standard. The comparison of the GPS-referenced frequency comb against an optical reference system with an outstanding short-term frequency stability of Δ f / f = 3 × 10 −15 at τ = 1 s enabled to directly resolve the short-term frequency instability of a single comb line that is limited by the frequency instability of the underlying GPSDO. Based on this comparison, a maximum relative Allan deviation of Δ f / f = 4.6 × 10 −12 at τ = 8s was obtained demonstrating the typical limitations of a GPS-referenced frequency comb that makes a frequency stability better than Δ f / f = 1 × 10 −12 only accessible for integration times longer than τ = 8000 s. Furthermore, the currently used He-Ne heterodyne source provides only a limited output power thus lowering the signal-to-noise ratio of the interferometer signals in comparison to the prior interferometer configuration with three independent He-Ne lasers. Our upcoming experiments will thus focus on a characterization of the frequency noise properties of the comb-referenced He-Ne heterodyne source with respect to comb line frequency noise and fiber dissemination to clarify if the high-frequency distortions of the comb line also enhance the frequency noise of the currently used He-Ne lasers. Future experiments will also have to enhance the currently limited output power of the GPS-referenced wavelength standard to feed all of the interferometer axes of the NPMM-200. Although the number of comb-referenced He-Ne lasers is easily scalable within the current control system, it is questionable if in terms of different noise properties of the He-Ne sources, the build-up of an extended laser array is desirable. Therefore, we will concentrate on directly integrating the ORS as an ultrastable, high-power laser source into the length measuring system of the NPMM-200. For this purpose, a suitable procedure for a correction of the cavity drift by means of the GPS-referenced frequency comb has to be established. 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The base plate below the metrology frame additionally carries the guiding and drive system of the x -, y -, and z -axis (see details in [ 32 , 40 ]). Figure 2. Scheme of a possible traceability chain for laser wavelength calibration deploying iodine-stabilized He-Ne lasers. The given uncertainties were taken from the literature as given within the text. Figure 3. Structure of the GPS-referenced frequency comb at the IPMS. The abbreviations denote: OFC—optical frequency comb, GPSDO—GPS disciplined oscillator, SDU—signal distribution unit, PD—photodiode. The numbers 1–4 correspond to the respective frequency counter channels. Figure 5. Frequency stability of the ORS as retrieved from manufacturer data (M.d.). ( a ) Time dependent beat frequency between two optical reference systems of comparable frequency stability with linear drift removed, ( b ) corresponding relative Allan deviation (see Section 2.4 ). Figure 6. Layout of the comb-referenced He-Ne heterodyne source. The abbreviations denote: GPSDO—GPS disciplined oscillator, SDU—signal distribution unit, APD—avalanche photodiode, PBS—polarizing beam splitter, M—mirror, BSA—beam splitter assembly, FI—Faraday isolator, FC—fiber coupling, UIC—transconductive amplifier (U/I converter), FPGA—field programmable gate array. Figure 7. Frequency instability of the free-running comb. ( a ) Time traces of the free-running comb parameters measured with the FXM50. The left y -axis depicts the change of repetition rate from its initial value. The right y -axis the CEO. ( b ) Time trace of the beat note between the respective comb line and the ORS. Since the frequency υ ORS of the ORS lies above the respective frequency of the comb line υ n , an increase in the repetition rate f Rep results in a decrease of the beat frequency f Beat . To monitor the frequency changes of the beat note, the measurements were performed without the bandpass filter at 60 MHz. Figure 8. In-loop comb parameters of the GPS-referenced frequency comb. ( a ) Time trace of the locked repetition rate. ( b ) Time trace of the locked CEO. Both plots depict the frequency deviations from the mean value over a measurement time of 24 h. ( c ) Calculated Allan deviations. The gray and blue lines present a linear fit to the data as described within the text. Figure 9. Frequency instability of an in-loop GPS-referenced frequency comb. ( a ) Time trace of the heterodyne signal between the OFC and the ORS. The red line indicates a linear fit to the data with f = 62.384 MHz–0.067 Hz/s. ( b ) A 1 h section of the frequency fluctuations of the heterodyne signal after the linear drift was removed from the measured data. ( c ) The respective relative Allan deviation of the heterodyne signal obtained by dividing the Allan deviation by f HeNe ≈ f Comb ≈ 474 THz. The abbreviation M.d. denotes “manufacturer data”. Figure 10. Frequency instability of the He-Ne heterodyne source used as a starting point for the realization of a comb-referenced laser module. ( a ) A 24 h-measurement series of the secondary standard laser (SSL) against a comb line, ( b ) 24 h measurement of the metrology laser (ML). In each of the plots, the frequency deviations from the mean value derived over the whole measurement series of 24 h are shown. ( c ) Relative Allan deviations obtained by dividing the frequency fluctuations by a nominal value of 474 THz. Figure 11. Frequency instability of the comb-referenced-He-Ne heterodyne source. ( a ) 24-h measurement series of the secondary standard laser (SSL) prior and after closing the control loop (closed-loop operation reproduced from [ 35 ]). ( b ) Relative Allan deviations. The Allan deviation of the OFC is obtained by combining the Allan deviation as calculated from the measurement of the beat signal between the ORS and the OFC for integration times below 4000 s and the Allan deviation given by the manufacturer for integration times above 4000 s. ( c ) The 1 h measurement sections of the 24-h timetrace. The upper graph depicts the SSL locked to the comb and the frequency fluctuations of the drift-removed beat signal between a comb line and the ORS (taken from Figure 9 b) for comparison. Please note that in contrast to the in-loop measurements of the heterodyne source, those measurements were not taken simultaneously. The graph in the middle enlarges the frequency scale to illustrate the frequency fluctuations of the SSL when locked to the comb line. The lower graph further enlarges the frequency scale to depict the fluctuations of the beat signal between the ML and the SSL, when locked to the comb line. All three graphs show the same 1 h section and present the frequency deviations from the respective mean value of the whole measurement series. Figure 12. Scheme of the reduced traceability chain for laser wavelength calibration of the metrology lasers of the NPMM-200. Figure 13. Influence of frequency distortions on the interferometric length measurement within the NPMM-200. ( a , b ) transform the frequency fluctuations and uncertainty of the comb line into a theoretical length measurement error for a maximum measurement range of L = 200 mm. ( c , d ) experimentally demonstrate the influence of frequency changes on the interferometric length measurement within the NPMM-200 at a constant distance of L = 100 mm using the comb-referenced He-Ne laser as a tunable laser source, ( c ) equidistant frequency steps of Δ f = 1.25 MHz and ( d ) repeated frequency jumps of Δ f = 0.625 MHz height. Integration time 0.5 s [ 35 ]. Chicago/Turabian Style Blumröder, Ulrike, Paul Köchert, Thomas Fröhlich, Thomas Kissinger, Ingo Ortlepp, Jens Flügge, Harald Bosse, and Eberhard Manske. 2023. "A GPS-Referenced Wavelength Standard for High-Precision Displacement Interferometry at λ = 633 nm" Sensors23, no. 3: 1734. https://doi.org/10.3390/s23031734 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here . Article Metrics Export citation file: BibTeX \| EndNote \| RIS MDPI and ACS Style Blumröder, U.; Köchert, P.; Fröhlich, T.; Kissinger, T.; Ortlepp, I.; Flügge, J.; Bosse, H.; Manske, E. A GPS-Referenced Wavelength Standard for High-Precision Displacement Interferometry at λ = 633 nm. Sensors 2023, 23, 1734. https://doi.org/10.3390/s23031734 AMA Style Blumröder U, Köchert P, Fröhlich T, Kissinger T, Ortlepp I, Flügge J, Bosse H, Manske E. A GPS-Referenced Wavelength Standard for High-Precision Displacement Interferometry at λ = 633 nm. Sensors. 2023; 23(3):1734. https://doi.org/10.3390/s23031734 Chicago/Turabian Style	https://www.mdpi.com/1424-8220/23/3/1734/xml
PUNJAB NATIONAL BANK (Constituted Under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 As Punjab National Bank on July 19, 1969 - DocsLib RED HERRING PROSPECTUS Dated February 14, 2005 100% Book Building Issue PUNJAB NATIONAL BANK (Constituted under the Banking Companies (Acquisition and Transfer PUNJAB NATIONAL BANK (Constituted Under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 As Punjab National Bank on July 19, 1969 - DocsLib sign in sign up PUNJAB NATIONAL BANK (Constituted Under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 As Punjab National Bank on July 19, 1969 [PDF] Related documentation The Institute of Cost Accountants of India Statement of Unpaid and Unclaimed Dividend Amount for FY 2018-19 Retail User Guide Sustainability and Ethical Banking: a Case Study of Punjab National Bank Corporate Overview Transact with Ease: Solutions That Work for Everyone, Everywhere Financial Inclusion-Account Opening Form Faqs Answers PUNJAB NATIONAL BANK RESOURCE MOBALISATION DIVISION HO: NEW DELHI FAQ's NRO Deposit Scheme 1. What Is NRO A/C? Sr.No . Ward Name Father/Husband Adhar Bank Name Accountno Canara Bank Mini Statement Toll Free Number PPFASMFSIP I-SIP Bank List 26.07.2021 List of Bank Names Contract Basis Job in Punjab National Bank Analyzing Punjab National Bank Scam Ashutosh Kolte* Assistant Professor, Department of Management Sciences, (PUMBA), Savitribai Phule Pune University A Case Study on Punjab National Bank Scam Telephone No. Mobile No. Email Address Contact (UPI): a Cashless Indian E-Transaction Process Punjab National Bank Account Statement Punjab National Bank Online Cheque Book Request Form Authorised Payment Channels of CESC Limited • Axis Bank Gateway Gold Investment Minted Products Issued by 10 Banks in India Equity Shareholding Pattern As on 30Th June 2021 List of General Managers of Banks Based on June'21 Results) Punjab National Bank (PNB (CIN) of the Company Not Applicable 2. Name Of Annex I List of State-Wise SLBC Convenor Banks and District-Wise Punjab National Bank Account Opening Form Lead District Manager (LDM) - Contact Details Name of S.No Name Address Phone No Mobile No MICR CODES of BANKS.Pdf List of Nodal Officers of Cppcs ( Dated 05.03.2020) Address CPPC PUNJAB NATIONAL BANK (Constituted Under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 As Punjab National Bank on July 19, 1969 Information Regarding Brick & Mortar Branches, BC/CSP Axis Bank Statement with Ifsc Code Master Debit Card Accidental Death Claim Form and Process Debit Card Request Pnb 1 Punjab National Bank REQUEST for PROPOSAL (RFP) Punjab National Bank Now India's Second Largest Public Sector Bank PUNJAB NATIONAL BANK Request for Proposal (RFP) A Study of Performance Appraisal and Dimensions of Organizational Climate in ICICI Bank and Punjab National Bank Sr. No. BIC CODE INSTITUTION NAME BRANCH INFORMATION CITY A Comparative Study of Icici Bank and Punjab National Bank To Send Money from Punjab National Bank (PNB) to Everest Bank Ltd, Nepal Via Indo Nepal Remittance Head Office : 7, Bhikaji Cama Place, New Delhi - 110066 List of Gst Registrations State-Wise/Union-Territory-Wise S.No Monetary Compensation Scheme for the Family of Active PNB Debit Country-Wise Branches of Indian Banks at Overseas Centres As on January 31, 2014 District Wise Consolidated List of 433 Bank Branches of PNB , JK Bank and YES Bank for Yatra 2017 LIST of Cppcs Cash Flow Statements of Punjab National Bank and ICICI Bank: a Comparative Analysis Page 4 UCO Bank's Rankings Most Important Terms and Conditions (MITC) Canara Bank Baddi Bank of India Mandi Punjab National Bank << Home , List of banks in Asia , Nedungadi Bank , Punjab National Bank RED HERRING PROSPECTUS Dated February 14, 2005 100% Book Building Issue PUNJAB NATIONAL BANK (Constituted under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 as Punjab National Bank on July 19, 1969. On April 13, 1987, the Head Office of our Bank was changed from 5, Sansad Marg, New Delhi 110 001 to 7, Bhikhaiji Cama Place, New Delhi 110 066). Head Office: 7, Bhikhaiji Cama Place, New Delhi 110 066, India . Tel: (91 11) 2610 2303; Fax (91 11) 2610 8741 E-mail: fpo@pnb.co.in ; Website: www.pnbindia.com PUBLIC ISSUE OF UP TO 80,000,000 EQUITY SHARES OF RS. 10 EACH FOR CASH AT A PRICE OF RS. [●] PER EQUITY SHARE AGGREGATING RS. [●] MILLION (THE “ISSUE”) BY PUNJAB NATIONAL BANK (“THE BANK” OR “THE ISSUER”). THE ISSUE COMPRISES A NET ISSUE TO THE PUBLIC OF UP TO 64,000,000 EQUITY SHARES OF RS. 10 EACH (THE “NET ISSUE”), A RESERVATION FOR EMPLOYEES OF UP TO 8,000,000 EQUITY SHARES OF RS. 10 EACH AND A RESERVATION FOR EXISTING SHAREHOLDERS OF UP TO 8,000,000 EQUITY SHARES OF RS. 10 EACH, AT THE ISSUE PRICE. THE ISSUE WOULD CONSTITUTE 25.37% OF THE FULLY DILUTED POST-ISSUE PAID-UP CAPITAL OF THE BANK AFTER GIVING EFFECT TO THE REDUCTION OF THE SHAREHOLDING OF THE PRESIDENT OF INDIA IN CONNECTION WITH THE CAPITAL REDUCTION AS DESCRIBED ON PAGE 22. PRICE BAND: RS. [●] TO RS. [●] PER EQUITY SHARE OF FACE VALUE RS. 10. THE PRICE BAND FOR THE ISSUE WILL BE DECIDED BY US IN CONSULTATION WITH THE BRLMS AND ADVERTISED AT LEAST ONE DAY PRIOR TO THE ISSUE OPENING DATE. WITH REGARD TO THE PRICE BAND/FLOOR PRICE, BIDDERS ARE ADVISED TO BE GUIDED BY THE MARKET PRICES OF OUR LISTED EQUITY SHARES. THE ISSUE PRICE IS [●] TIMES OF THE FACE VALUE AT THE LOWER END OF THE PRICE BAND AND [●] TIMES OF THE FACE VALUE AT THE HIGHER END OF THE PRICE BAND In case of revision in the Price Band, the Bidding Period will be extended for three additional days after revision of the Price Band subject to the Bidding Period/Issue Period not exceeding 13 days. Any revision in the Price Band and the revised Bidding Period, if applicable, will be widely disseminated by notification to the National Stock Exchange of India Limited and the Stock Exchange, Mumbai , by issuing a press release, and also by indicating the change on the websites of the Book Running Lead Managers (“BRLMs”) and at the terminals of the Syndicate. The Issue is being made through the 100% Book Building Process wherein up to 50% of the Net Issue to the public shall be allocated on a discretionary basis to Qualified Institutional Buyers. Further, not less than 15% of the Net Issue to the public shall be available for allocation on a proportionate basis to Non- Institutional Bidders and not less than 35% of the Net Issue to the public shall be available for allocation on a proportionate basis to Retail Individual Bidders, subject to valid Bids being received at or above the Issue Price. Up to 8,000,000 Equity Shares shall be available for allocation on a proportionate basis to the Employees, subject to valid Bids being received at or above the Issue Price and the maximum Bid in this portion is limited to 500 Equity Shares. Up to 8,000,000 Equity Shares shall be available for allocation on a proportionate basis to the Existing Shareholders, subject to valid Bids being received at or above the Issue Price. RISK IN RELATION TO THE ISSUE The Issue Price (as determined by Punjab National Bank in consultation with the Book Running Lead Managers on the basis of assessment of market demand for the Equity Shares by way of book-building) should not be taken to be indicative of the market price of the Equity Shares after the Equity Shares are listed. The market price of the existing Equity Shares of Punjab National Bank could affect the price discovery through book building and vice versa. No assurance can be given regarding an active and/or sustained trading in the Equity Shares or regarding the price at which the Equity Shares will be traded after listing. GENERAL RISKS Investments in equity and equity-related securities involve a degree of risk and investors should not invest any funds in this Issue unless they can afford to take the risk of losing their investment. Investors are advised to read the risk factors carefully before taking an investment decision in this Issue. For taking an investment decision, investors must rely on their own examination of the Bank and the Issue including the risks involved. The Equity Shares offered in the Issue have not been recommended or approved by the Securities and Exchange Board of India (“SEBI”), nor does SEBI guarantee the accuracy or adequacy of this Red Herring Prospectus. Specific attention of the investors is invited to the section titled “Risk Factors” beginning on page x. ISSUER’S ABSOLUTE RESPONSIBILITY The Bank having made all reasonable inquiries, accepts responsibility for and confirms that this Red Herring Prospectus contains all information with regard to the Bank and the Issue, which is material in the context of the Issue, that the information contained in this Red Herring Prospectus is true and correct in all material aspects and is not misleading in any material respect, that the opinions and intentions expressed herein are honestly held and that there are no other facts, the omission of which makes this Red Herring Prospectus as a whole or any of such information or the expression of any such opinions or intentions misleading in any material respect. LISTING ARRANGEMENT The Equity Shares offered through this Red Herring Prospectus are proposed to be listed on the National Stock Exchange of India Limited and the Stock Exchange, Mumbai, where the existing Equity Shares of the Bank are listed. We have received in-principle approval from the National Stock Exchange of India Limited and the Stock Exchange, Mumbai for the listing of our Equity Shares pursuant to letters dated January 18, 2005 and January 28, 2005, respectively. BOOK RUNNING LEAD MANAGERS ICICI SECURITIES LIMITED DSP MERRILL LYNCH LIMITED ENAM FINANCIAL JM MORGAN STANLEY ICICI Centre, Mafatlal Centre, 10th Floor, CONSULTANTS PVT LTD PRIVATE LIMITED H.T.Parekh Marg, Churchgate Nariman Point, 801/ 802, Dalamal Towers, 141, Maker Chambers III, Mumbai 400 020, India Mumbai 400 021, India Nariman Point, Nariman Point, Tel : (91 22) 2288 2460 Tel: (91 22) 226 21071 Mumbai 400 021, India Mumbai 400 021, India Fax : (91 22) 2282 6580 Fax: (91 22) 226 21187 Tel : (91 22) 5638 1800 Tel: (91 22) 1600 22 0004/5630 3030 E-mail : pnb_fpo@isecltd.com E-mail: pnb_ fpo@ml.com Fax: (91 22) 2284 6824 Fax: (91 22) 5630 1694 E-mail: pnb.fpo@enam.com E-mail: pnbfpo@jmmorganstanley.com REGISTRAR TO THE ISSUE ISSUE PROGRAMME KOTAK MAHINDRA CAPITAL MCS LIMITED COMPANY LIMITED Sri Padmavati Bhavan BID / ISSUE OPENS ON : MONDAY, MARCH 7, 2005 rd Bakhtawar, 3 Floor, Plot No. 93, Road No. 16, 229, Nariman Point, MIDC Area, Andheri (East) Mumbai 400 021, India Mumbai 400 092 Tel: (91 22) 5634 1100 Tel: (91 22) 2820 1785 BID / ISSUE CLOSES ON : FRIDAY, MARCH 11, 2005 Fax: (91 22) 2284 0492 Fax: (91 22) 2820 1783 E-mail: pnb.fpo@kotak.com Email: pnbfpo@mcsind.com TABLE OF CONTENTS DEFINITIONS AND ABBREVIATIONS i CERTAIN CONVENTIONS; USE OF MARKET DATA viii FORWARD-LOOKING STATEMENTS ix RISK FACTORS x SUMMARY 1 SUMMARY FINANCIAL INFORMATION 3 THE ISSUE 6 GENERAL INFORMATION 7 CAPITAL STRUCTURE 22 OBJECTS OF THE ISSUE 29 THE INDIAN BANKING SECTOR 31 BUSINESS 39 HISTORY AND CERTAIN CORPORATE MATTERS 62 SELECTED FINANCIAL INFORMATION 67 SELECTED STATISTICAL INFORMATION 72 MANAGEMENT’S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS 90 RECENT DEVELOPMENTS 117 OUR MANAGEMENT 120 OUR PROMOTER, SUBSIDIARIES AND GROUP COMPANIES 127 RELATED PARTY TRANSACTIONS 149 REGULATIONS AND POLICIES 151 GOVERNMENT APPROVALS 182 OUTSTANDING LITIGATION AND MATERIAL DEVELOPMENTS 185 DIVIDEND POLICY 208 OTHER REGULATORY DISCLOSURES 209 STATEMENT OF TAX BENEFITS 211 FINANCIAL INDEBTEDNESS 216 TERMS OF THE ISSUE 217 ISSUE STRUCTURE 220 ISSUE PROCEDURE 223 BASIS FOR ISSUE PRICE 241 STATUTORY AND OTHER INFORMATION 243 MAIN PROVISIONS OF CONSTITUTIONAL DOCUMENTS 248 FINANCIAL STATEMENTS F-1 SUMMARY OF SIGNIFICANT DIFFERENCES BETWEEN INDIAN GAAP AND U.S. GAAP 270 MATERIAL CONTRACTS AND DOCUMENTS FOR INSPECTION 278 DECLARATION 280 DEFINITIONS AND ABBREVIATIONS DEFINITIONS Term Description “PNB” or “the Bank” or “our Bank” Punjab National Bank, a corresponding new bank constituted under the Banking or “Punjab National Bank ” Companies (Acquisition and Transfer of Undertakings) Act, 1970, and having its Head Office at 7, Bhikhaiji Cama Place, New Delhi 110 066, India. “we” or “us” and “our” Refers to Punjab National Bank and, where the context requires, its subsidiaries, which are PNB Housing Finance Limited, PNB Gilts Limited and PNB AMC Limited. Issue Related Terms Term Description Allotment Unless the context otherwise requires, the issue and allotment of Equity Shares, pursuant to this Issue. Auditors The statutory auditors of the Bank who are M/s. Surendar K. Jain & Co., Chartered Accountants, M/s. Mookherjee Biswas & Pathak, Chartered Accountants, M/s. M.C. Bhandari & Co., Chartered Accountants, M/s. P.K. Chopra & Co., Chartered Accountants, M/s. Ramanlal G. Shah & Co., Chartered Accountants and M/s. B.K. Ramadhyani & Co., Chartered Accountants. Bank Acquisition Act Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970, as amended from time to time. Bank Regulations Punjab National Bank (Shares & Meetings) Regulations, 2000, as amended from time to time, which have been formulated by the Board of Directors in the exercise of powers conferred by Section 19 of the Bank Acquisition Act after consultation with the RBI and with previous sanction of the Government. Bank Scheme The Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970, notified under Section 9 of the Bank Acquisition Act. Banker to the Issue Punjab National Bank. Banking Regulation Act The Banking Regulation Act, 1949, as amended from time to time. Bid An indication to make an offer during the Bidding Period by a prospective investor to subscribe to our Equity Shares at a price within the Price Band, including all revisions and modifications thereto. Bid Amount The highest value of the optional Bids indicated in the Bid cum Application Form and payable by the Bidder on submission of the Bid in the Issue. Bid Closing Date /Issue Closing Date The date after which the Syndicate will not accept any Bids for the Issue, which shall be notified in an English language newspaper with wide circulation and a Hindi language newspaper with wide circulation. Bid cum Application Form The form in terms of which the Bidder shall make an offer to subscribe to the Equity Shares and which will be considered as the application for Allotment of the Equity Shares pursuant to the terms of this Red Herring Prospectus. Bid Opening Date/Issue Opening Date The date on which the Syndicate shall start accepting Bids for the Issue, which shall be the date notified in an English language newspaper and a Hindi language newspaper with wide circulation. i Bidder Any prospective investor who makes a Bid pursuant to the terms of this Red Herring Prospectus and the Bid cum Application Form. Bidding Period/Issue Period The period between the Bid Opening Date/Issue Opening Date and the Bid Closing Date/Issue Closing Date, inclusive of both days, and during which prospective Bidders can submit their Bids. Board of Directors/Board The board of directors of our Bank or a committee constituted thereof. Book Building Process Book building route as provided under Chapter XI of the SEBI Guidelines, in terms of which the Issue is made. BRLMs/Book Running Lead Managers Book Running Lead Managers to the Issue, in this case being ICICI Securities Limited, DSP Merrill Lynch Limited, Enam Financial Consultants Private Limited, JM Morgan Stanley Private Limited and Kotak Mahindra Capital Company Limited. CAN/Confirmation of Allocation Note Means the note or advice or intimation of allocation of Equity Shares sent to the Bidders who have been allocated Equity Shares after discovery of the Issue Price in accordance with the Book Building Process. Capital Reduction As a condition of the Government Approval, the reduction of 30,000,000 Equity Shares held by the President of India in the Bank as described in the section titled “Capital Structure” on page 22. Cap Price The higher end of the Price Band, above which the Issue Price will not be finalized and above which no Bids will be accepted. Companies Act The Companies Act, 1956 as amended from time to time. Constitutional Documents The Bank Acquisition Act, the Bank Regulations and the Bank Scheme. Crore 10 million Cut-off Price Any price within the Price Band (notified at least one day prior to the Bid Opening Date/Issue Opening Date in Business Standard, an English language newspaper with wide circulation and Hindustan, a Hindi language newspaper with wide circulation) finalised by us in consultation with the BRLMs. A Bid submitted at Cut-off Price is a valid Bid at all price levels within the Price Band. DSE The Delhi Stock Exchange Association Limited. Depositories Act The Depositories Act, 1996, as amended from time to time. Depository A body corporate registered under the SEBI (Depositories and Participant) Regulations, 1996, as amended from time to time. Depository Participant A depository participant as defined under the Depositories Act. Designated Date The date on which funds are transferred from the Escrow Account(s) to the Issue Account after the Prospectus is filed with the Designated Stock Exchange, following which the Allotment will be made to successful Bidders. Designated Stock Exchange National Stock Exchange of India Limited. Director(s) Director(s) of Punjab National Bank, unless otherwise specified. Eligible NRI An NRI who is a resident of Bahrain , Kuwait or the United Arab Emirates . ii Employee All or any of the following: (a) a permanent employee of the Bank working in India as of January 1, 2005; (b) a director of the Bank, whether a whole time director or otherwise as of January 1, 2005. Employees Reservation Portion The portion of the Issue being a maximum of 8,000,000 Equity Shares available for allocation to Employees. Equity Shares Equity shares of the Bank of Rs. 10 each unless otherwise specified in the context thereof. Escrow Account The account opened with the Escrow Collection Bank and in whose favour the Bidder will issue cheques or drafts in respect of the Bid Amount when submitting a Bid. Escrow Agreement The agreement entered into between the Bank, the Registrar, the Escrow Collection Bank and the BRLMs for collection of the Bid Amounts and for remitting refunds, if any, of the amounts collected, to the Bidders. Escrow Collection Bank Punjab National Bank. Existing Shareholders Holders of Equity Shares as of February 11, 2005 worth up to Rs.100,000, determined on the basis of the closing price of the Equity Shares in the NSE on February 10, 2005. Existing Shareholders The portion of the Issue being a maximum of 8,000,000 Equity Shares available Reservation Portion for allocation to Existing Shareholders. Financial Year /fiscal year/ fiscal / FY The period of twelve months ended March 31 of that particular year, unless otherwise stated. First Bidder The Bidder whose name appears first in the Bid cum Application Form or Revision Form. Floor Price The lower end of the Price Band, below which the Issue Price will not be finalised and below which no Bids will be accepted. GoI/Government The Government of India . Government Approval The approval letter received by the Bank from the Department of Economic Affairs, MoF, GoI, bearing number F.No.11/29/2004-BOA, dated January 7, 2005, granting permission for the Issue subject to the conditions stated therein. Head Office The head office of the Bank being 7, Bhikhaiji Cama Place, New Delhi 110 066, India. I.T. Act The Income Tax Act, 1961, as amended from time to time. Indian GAAP Generally accepted accounting principles in India. Issue The public issue by the Bank of up to 80,000,000 Equity Shares for cash at the Issue Price of Rs. [●] per Equity Share aggregating Rs. [●] million and comprising a Net Issue to the public of up to 64,000,000 Equity Shares, a reservation for Employees of up to 8,000,000 Equity Shares and a reservation for Existing Shareholders of up to 8,000,000 Equity Shares. Issue Account Account opened with the Banker to the Issue to receive monies from the Escrow Accounts on the Designated Date. iii Issue Price The final price at which Equity Shares will be allotted in terms of the Red Herring Prospectus, as determined by the Bank in consultation with the BRLMs, on the Pricing Date. Lac/Lakh 0.1 million Margin Amount The amount paid by the Bidder at the time of submission of his/her Bid, being 0% to 100% of the Bid Amount. NRI/Non-Resident Indian Non-Resident Indian, is a Person resident outside India, who is a citizen of India or a Person of Indian origin and shall have the same meaning as ascribed to such term in the Foreign Exchange Management (Transfer or Issue of Security by a Person Resident Outside India) Regulations, 2000. NSE National Stock Exchange of India Limited. Nedungadi Bank Nedungadi Bank Ltd., which was merged with PNB in fiscal 2003. Net Issue/Net Issue to the public The Issue less the allocation to the Employees and Existing Shareholders. Non-Institutional Bidders All Bidders that are not eligible Qualified Institutional Buyers for this Issue, including affiliates of BRLMs and Syndicate Members, or Retail Individual Bidders and who have Bid for an amount more than Rs. 100,000. For the sake of clarity NRIs, other than Eligible NRIs, foreign venture capital investors and Indian venture capital funds are not included within this definition. Non-Institutional Portion The portion of the Net Issue to the public and being a minimum of 9,600,000 Equity Shares available for allocation to Non-Institutional Bidders. Non Residents Non-Resident is a Person resident outside India, as defined under FEMA and who is a citizen of India or a Person of Indian Origin under Foreign Exchange Management (Transfer or Issue of Security by a Person Resident Outside India) Regulations, 2000. OCB/Overseas Corporate Body A company, partnership, society or other corporate body owned directly or indirectly to the extent of at least 60% by NRIs including overseas trusts, in which not less than 60% of beneficial interest is irrevocably held by NRIs directly or indirectly as defined under Foreign Exchange Management (Transfer or Issue of Security by a Person Resident Outside India) Regulations, 2000. PIO/ Person of Indian Origin Shall have the same meaning as is ascribed to such term in the Foreign Exchange Management (Investment in Firm or Proprietary Concern in India) Regulations, 2000. Pay-in Date Bid Closing Date or the last date specified in the CAN sent to Bidders, as applicable. Pay-in-Period (i) With respect to Bidders whose Margin Amount is 100% of the Bid Amount, the period commencing on the Bid Opening Date and extending until the Bid Closing Date, and (ii) with respect to Bidders whose Margin Amount is less than 100% of the Bid Amount, the period commencing on the Bid Opening Date and extending until the closure of the Pay-in Date, as specified in the CAN. Person/Persons Any individual, sole proprietorship, unincorporated association, unincorporated organization, body corporate, corporation, company, partnership, limited liability company, joint venture, or trust or any other entity or organization validly constituted and/or incorporated in the jurisdiction in which it exists and operates, as the context requires. iv Price Band The price band with a minimum price (Floor Price) of Rs. [●] and the maximum price (Cap Price) of Rs. [●], which shall be advertised at least one day prior to the Bid Opening Date/Issue Opening Date, in Business Standard, an English language newspaper with wide circulation and Hindustan, a Hindi language newspaper with wide circulation, and includes any revisions thereof. Pricing Date The date on which the Bank in consultation with the BRLMs finalises the Issue Price. Promoter The President of India acting through the MoF, GoI. Prospectus The Prospectus, to be filed with the Designated Stock Exchange containing, inter alia, the Issue Price that is determined at the end of the Book Building Process, the size of the Issue and certain other information. QIB Portion The portion of the Net Issue to public and up to 32,000,000 Equity Shares of Rs. 10 each at the Issue Price, available for allocation to QIBs. Qualified Institutional Buyers or QIBs Public financial institutions as specified in Section 4A of the Companies Act, FIIs, scheduled commercial banks, mutual funds registered with SEBI, multilateral and bilateral development financial institutions, state industrial development corporations, insurance companies registered with the Insurance Regulatory and Development Authority, provident funds with minimum corpus of Rs. 250 million (subject to applicable law) and pension funds with minimum corpus of Rs. 250 million. For the sake of clarity, foreign venture capital funds and Indian venture capital funds are not included within this definition. RHP or Red Herring Prospectus Means the document issued in accordance with the SEBI Guidelines, which does not have complete particulars of the Price Band, the Issue Price or the size of the Issue, which will be filed with the Designated Stock Exchange at least three days before the Bid Opening Date/Issue Opening Date and which will become a Prospectus after determination of Issue Price and upon filing with the Designated Stock Exchange. Registrar /Registrar to the Issue Registrar to the Issue, in this case being MCS Limited, having its office in Sri Padmavati Bhavan, Plot No. 93, Road No. 16, MIDC Area, Andheri (East), Mumbai 400 092. Reservation Portion The portion of the Issue being a maximum of 16,000,000 Equity Shares available for allocation to Employees and Existing Shareholders. Retail Individual Bidders Individual Bidders (including HUFs) who have bid for Equity Shares for an amount less than or equal to Rs. 100,000, in any of the bidding options in the Issue. For the sake of clarity, NRIs, other than Eligible NRIs, cannot participate in this Issue and bid for Equity Shares. Return of Proceeds The return by the Bank to the Government of the amount raised from the Issue of 30,000,000 Equity Shares in this Issue as required by the condition of the Government Approval as described on page 182. Retail Portion The portion of the Net Issue to the public and being a minimum of 22,400,000 Equity Shares of Rs.10 each available for allocation to Retail Individual Bidders. Revision Form The form used by the Bidders to modify the quantity of Equity Shares or the Bid price in any of their Bid cum Application Forms or any previous Revision Form(s). SEBI Act Securities and Exchange Board of India Act, 1992, as amended from time to time. v SEBI Guidelines SEBI (Disclosure and Investor Protection) Guidelines, 2000 issued by SEBI on January 27, 2000, as amended, and to the extent applicable to this Issue, including instructions and clarifications issued by SEBI from time to time. SEBI Takeover Regulations Securities and Exchange Board of India (Substantial Acquisition of Shares and Takeover) Regulations, 1997, as amended from time to time. Stock Exchanges BSE and NSE. Syndicate The BRLMs and the Syndicate Members. Syndicate Agreement The agreement to be entered into among the Bank and the members of the Syndicate, in relation to the collection of Bids in this Issue. Syndicate Members ICICI Brokerage Services Limited, DSP Merrill Lynch Limited, Enam Securities Private Limited, JM Morgan Stanley Retail Services Private Limited, and Kotak Securities Limited. Tier 1 capital The core capital of a bank. It comprises paid-up capital and reserves consisting of any statutory reserves, free reserves and capital reserves as reduced by equity investments in subsidiaries, intangible assets, and losses in the current period and those brought forward from the previous period. Tier 2 capital The undisclosed reserves and cumulative perpetual preference shares, revaluation reserves (at a discount of 55.0%), general provisions and loss reserves (allowed up to a maximum of 1.25% of risk weighted assets), hybrid debt capital instruments (which combine certain features of both equity and debt securities) and subordinated debt. Tier 2 Bonds or Subordinated Bonds Unsecured subordinated bonds issued by PNB for Tier 2 capital adequacy purposes. TRS or Transaction Registration Slip The slip or document issued by the Syndicate Members to the Bidder as proof of registration of the Bid. U.S. GAAP Generally accepted accounting principles in the United States of America. Underwriters The BRLMs and the Syndicate Members. Underwriting Agreement Agreement among the members of the Syndicate and the Bank to be entered into on or after the Pricing Date. ABBREVIATIONS Abbreviation Full Form AS Accounting Standards as issued by the Institute of Chartered Accountants of India BIFR The Board of Industrial and Financial Reconstruction BSE The Stock Exchange, Mumbai CAGR Compounded Annual Growth Rate CAIIB Certified Associate of Indian Institute of Bankers CDSL Central Depository Services (India) Limited CRR Cash Reserve Ratio vi DI Derecognised Interest DRT Debt Recovery Tribunal DSPML DSP Merrill Lynch Limited EGM Extraordinary General Meeting ENBI Erstwhile New Bank of India Enam Enam Financial Consultants Private Limited EPS Earnings per share FCNR Account Foreign Currency Non Resident Account FEMA Foreign Exchange Management Act, 1999, as amended from time to time, and the regulations framed thereunder FII Foreign Institutional Investor (as defined under Foreign Exchange Management (Transfer or Issue of Security by a Person Resident outside India) Regulations, 2000) registered with SEBI under applicable laws in India FIPB Foreign Investment Promotion Board GIR Number General Index Registry Number HUF Hindu Undivided Family IPO Initial Public Offering ISEC ICICI Securities Limited JMMS JM Morgan Stanley Private Limited Kotak Kotak Mahindra Capital Company Limited LC Letters of Credit MoF Ministry of Finance, Government of India NAV Net Asset Value NPA Non Performing Asset NRE Account Non Resident External Account NRO Account Non Resident Ordinary Account NSDL National Securities Depository Limited P/E Ratio Price/Earnings Ratio PAN Permanent Account Number PSU Public Sector Undertaking RBI The Reserve Bank of India RoNW Return on Net Worth SCRR Securities Contracts (Regulation) Rules, 1957, as amended from time to time SEBI The Securities and Exchange Board of India constituted under the SEBI Act, 1992 SI Suspended Interest SIA Secretariat for Industrial Assistance SLR Statutory Liquidity Ratio Supreme Court Hon’ble Supreme Court of India vii CERTAIN CONVENTIONS; USE OF MARKET DATA Unless stated otherwise, the financial data in this Red Herring Prospectus is derived from our unconsolidated financial statements prepared in accordance with Indian GAAP and included in this Red Herring Prospectus. Unless stated otherwise, the statistical and operational data in this Red Herring Prospectus is presented on an unconsolidated basis. Our fiscal year commences on April 1 and ends on March 31 of each year, so all references to a particular fiscal year are to the twelve-month period ended March 31 of that year. The six months ended September 30, 2003 and 2004 are referred to as the first six months of fiscal 2004 and the first six months of fiscal 2005, respectively. In this Red Herring Prospectus, any discrepancies in any table between the total and the sums of the amounts listed are due to rounding. There are significant differences between Indian GAAP and U.S. GAAP; accordingly, the degree to which the Indian GAAP financial statements included in this Red Herring Prospectus will provide meaningful information is entirely dependent on the reader’s level of familiarity with Indian accounting practices. Any reliance by Persons not familiar with Indian accounting practices on the financial disclosures presented in this Red Herring Prospectus should accordingly be limited. We have not attempted to explain those differences or quantify their impact on the financial data included herein, and we urge you to consult your own advisors regarding such differences and their impact on our financial data. All references to “India” contained in this Red Herring Prospectus are to the Republic of India. All references to “Rupees” or “Rs.” are to Indian Rupees, the official currency of the Republic of India. All references to “US$”; “U.S. Dollar” or “US Dollars” are to United States Dollars, the official currency of the United States of America. For additional definitions, please see the section titled “Definitions and Abbreviations” on page i. Unless stated otherwise, industry data used throughout this Red Herring Prospectus has been obtained from RBI publications and other industry sources. Industry publications generally state that the information contained in those publications has been obtained from sources believed to be reliable but that their accuracy and completeness are not guaranteed and their reliability cannot be assured. Although we believe that industry data used in this Red Herring Prospectus is reliable, it has not been independently verified. viii FORWARD-LOOKING STATEMENTS We have included statements in this Red Herring Prospectus which contain words or phrases such as “will”, “aim”, “will likely result”, “believe”, “expect”, “will continue”, “anticipate”, “estimate”, “intend”, “plan”, “contemplate”, “seek to”, “future”, “objective”, “goal”, “project”, “should”, “will pursue” and similar expressions or variations of such expressions, that are “forward- looking statements”. All forward-looking statements are subject to risks, uncertainties and assumptions that could cause actual results to differ materially from those contemplated by the relevant forward-looking statement. Important factors that could cause actual results to differ materially from our expectations include, among others: ● General economic and business conditions in India and other countries; ● Our ability to successfully implement our strategy, growth and expansion plans and technological initiatives; ● Changes in Indian or international interest rates and their impact on our financial results; ● Performance of the agricultural, retail and industrial sectors in India; ● Rate of growth of our deposits, advances and investments; ● Changes in the value of the Rupee and other currencies; ● Potential mergers, acquisitions or restructurings; ● Changes in laws and regulations that apply to banks in India, including laws that impact our ability to enforce our collateral; ● The occurrence of natural disasters or calamities affecting the areas in which we have operations or outstanding credit; ● Changes in political conditions in India; and ● Changes in the foreign exchange control regulations in India. For further discussion of factors that could cause our actual results to differ, see the section titled “Risk Factors” beginning on page x. By their nature, certain market risk disclosures are only estimates and could be materially different from what actually occurs in the future. As a result, actual future gains or losses could materially differ from those that have been estimated. The Bank, the members of the Syndicate and their respective affiliates do not have any obligation to, and do not intend to, update or otherwise revise any statements reflecting circumstances arising after the date hereof or to reflect the occurrence of underlying events, even if the underlying assumptions do not come to fruition. In accordance with SEBI requirements, the Bank and the BRLMs will ensure that investors in India are informed of material developments until such time as the grant of listing and trading permission by the Stock Exchanges in respect of the Equity Shares Allotted in this Issue. ix RISK FACTORS An investment in equity shares involves a high degree of risk. You should carefully consider all the information in this Red Herring Prospectus, including the risks and uncertainties described below, before making an investment in our Equity Shares. If the following risks actually occur, our business, results of operations and financial condition could suffer, and the price of our Equity Shares and the value of your investment in our Equity Shares could decline. Internal Risk Factors and Risks relating to Our Business Our results of operations depend to a great extent on our net interest income, and volatility in interest rates and other market conditions could adversely impact our business and financial results. In the first six months of fiscal 2005, net interest income represented 46.0% of our interest income and 36.7% of our total income. Changes in market interest rates could affect the interest we earn on our assets differently from the interest we pay on our liabilities. The difference could result in an increase in interest expense relative to interest income leading to a reduction in net interest income. Under the regulations of the RBI, we are required to maintain a minimum specified percentage, currently 25.0%, of our net demand and time liabilities, in government or other approved securities. As of September 30, 2004, 38.2% of our net demand and time liabilities and 84.0% of our total investments were in these securities. Yields on these investments, as well as yields on our other interest earning assets, are dependent to a large extent on interest rates. Interest rates are sensitive to many factors beyond our control, including the RBI’s monetary policies, deregulation of the financial sector in India, domestic and international economic and political conditions and other factors. Any volatility in interest rates could adversely affect our business and financial performance. On October 26, 2004, the RBI increased the reverse repo rate, which is the rate at which commercial banks lend funds to the RBI, from 4.50% to 4.75%. In a rising interest rate environment, especially if the rise were sudden or sharp, we could be materially adversely affected by the decline in the market value of our government securities portfolio and other fixed income securities and may be required to realize mark to market losses. Additionally, an increase in interest rates may harm the retail and corporate sectors, which may adversely impact our business. We could be subject to volatility in income from our treasury and trading operations. Approximately 51.0% of our total income in fiscal 2004 and approximately 43.4% of our total income in the first half of fiscal 2005 was generated by our treasury division. Additionally, we have entered or increased our operations in other trading related businesses such as gold, foreign exchange and derivatives. Our treasury and trading operations are vulnerable to changes in interest rates, exchange rates, equity prices and other factors. In particular, if interest rates rise we may not be able to realize the same level of income from treasury operations as we have in the past. Any decrease in our income due to volatility in income from these activities could adversely affect our business. x If we are not able to reduce or maintain the level of non-performing assets in our portfolio, our business and financial condition may be adversely affected. Our gross non-performing assets were Rs. 41.5 billion representing 7.65% of our gross advances as of September 30, 2004 and Rs. 46.7 billion representing 9.35% of our gross advances as of March 31, 2004. Our net non-performing assets represented 0.30% of our net advances as of September 30, 2004 and 0.98% of our net advances as of March 31, 2004. Our ten largest gross non-performing assets constituted 13.5% of our gross non-performing assets as of September 30, 2004. Information regarding our ten largest non-performing assets is provided in the section titled “Selected Statistical Information” on page 72. Although we have been able to reduce our net non-performing assets through recoveries, upgrading the classification of assets into “performing” categories and additional provisioning, we cannot assure you that we will be able to maintain this level of non-performing assets in the future or that we will be able to reduce, or prevent an increase in, our gross or net non-performing assets. A number of factors which are not within our control could affect our ability to control and reduce non-performing assets, including increased competition arising from economic liberalization in India, variable industrial and agricultural growth, declines in commodity and food grain prices, the high level of debt in the capital structures of Indian companies, fluctuations in interest and exchange rates, changes in regulations, including with respect to directed lending, and the high interest rates in the Indian economy during the period in which a large number of borrowers made their borrowings. In addition, the expansion of our business and our entry into new businesses may cause our non-performing loans to increase and the overall quality of our loan portfolio to deteriorate. As of September 30, 2004, approximately 45.3% of our gross non-performing asset portfolio was concentrated in the following sectors: iron and steel (16.0%), textiles (15.9%), chemicals, dyes and paints (5.5%) and engineering (7.9%). These sectors together constituted 14.3% of our total funded credit exposure to accounts larger than Rs. 10 million as of September 30, 2004. These sectors have been adversely affected by economic conditions in recent years in varying degrees. Although our loan portfolio contains loans to a wide variety of businesses, financial difficulties in these industries could increase our level of non-performing assets and adversely affect our business and financial condition. We have sold part of our non-performing assets to an asset reconstruction company, Asset Reconstruction Company (India) Limited (“ARCIL”), against security receipts issued by ARCIL. As of September 30, 2004, we had investments of Rs. 303.7 million in security receipts issued by ARCIL. The price at which the assets were sold was determined on an arms length basis and the sale has resulted in the removal of the non-performing assets from our books. The recovery of principal and the rate of return on the investments in security receipts are dependent on the recovery of the underlying assets. These investments in security receipts are required to be valued at the net asset value as declared by ARCIL. There can be no assurance that ARCIL will be able to recover these assets and we will be able to redeem our investments or that there will be no reduction in the value of these investments. xi We are required to return the Issue Proceeds corresponding to the issue of 30,000,000 Equity Shares to the Government by March 31, 2005. One of the conditions for the Government Approval for the Issue requires us to return to the Government a sum corresponding to the Issue Proceeds relating to the issue of 30,000,000 Equity Shares by March 31, 2005. As a result, after the payment of the aforesaid sum to the Government, the shareholding of the President of India in our Bank will be reduced by 30,000,000 Equity Shares. For further details see the section title “Capital Structure” on page 22. The Return of Proceeds to the Government and the Capital Reduction could have adverse tax or other consequences for us. Although the Bank Acquisition Act permits us to reduce the share capital of the Government, there is some ambiguity as to whether it permits such reduction from the share premium account. Although we believe that the Bank Acquisition Act permits such reduction to be made from the share premium account and we have obtained independent opinions to such effect, in the event the Bank Acquisition Act were interpreted differently by statutory authorities, it is possible that the part of the payment to the Government which represents the amount debited from the share premium account could be deemed to be a dividend and consequently, there could be tax implications for us. We have obtained independent opinions that the payment should not be deemed to be a dividend and should not in any event be subject to any dividend tax. We have also requested that the Government facilitate a waiver of any such tax implications on the Bank or indemnify the Bank for the same. However, we have, as of the date hereof, not received a response from the Government and neither the Issue nor the Capital Reduction is conditioned on receipt of such a response. The standard tax imposed on a bank in respect of a dividend paid is 12.5% of the amount of the dividend, plus a surcharge of 2.5% and on the total, a further 2% education cess, although the tax authorities have the right to seek or impose additional penalties and other amounts in certain circumstances under applicable laws. In the event that the Bank Acquisition Act is interpreted so as not to permit the payment to the Government from the share premium account, and the Government does not respond in a satisfactory manner to our request, we could suffer tax or other consequences arising from the characterization of the transaction as to which we are not indemnified, and such consequences could be material. For further details on the mechanism, modalities and impact of the Capital Reduction please see the sections titled “General Information” on page 7, “Capital Structure” on page 22 and “Objects of the Issue” on page 29. The independent opinions on tax and the letter dated February 7, 2005 written by our Bank to the Government are available for public inspection. See the section titled “Material Contracts and Documents for Inspection” on page 278. We have substantial activity in the agriculture and other priority sectors and therefore our business could be adversely affected by market and other factors that impact these sectors. The directed lending norms of the RBI require that every bank should extend at least 40% of its net xii bank credit to certain eligible sectors, such as agriculture and small-scale industries, which are categorized as “priority sectors”. As of the last reporting Friday of September 2004, priority sector credit (including Rural Infrastructure Development Fund (“RIDF”)) constituted 47.7% of our net bank credit, and loans to agricultural and small scale industry borrowers constituted 18.8% and 11.3%, respectively, of our net bank credit. As of September 30, 2004, the percentage of our priority sector gross non-performing assets to total priority sector advances was 8.03%. As of September 30, 2004, the percentage of our agricultural gross non-performing assets to total agricultural advances was 4.37% and the percentage of our small scale industry gross non-performing assets to total small scale industry advances was 16.53%, compared to our overall percentage of non-performing assets to total advances of 7.65%. In addition, as of the last reporting Friday of September, 72.5% of our agricultural loan portfolio was concentrated in northern India. Economic difficulties owing to various factors, such as uneven monsoons or other weather conditions, natural calamities and reductions in price supports or other changes in government policy, or other events and conditions, are likely to adversely impact these priority sectors and therefore our business and level of non-performing assets. We may be unable to sustain the growth rate of our retail loan business. We have achieved significant growth in our retail loan business in recent years. Between March 31, 2002 and March 31, 2004, our average volume of retail advances grew at a compound annual growth rate of 53.2%. As of September 30, 2004, retail loans represented 19.2% of our total outstanding credit as compared to 19.4% and 16.3% of our total outstanding credit as of March 31, 2004 and March 31, 2003, respectively. Our present business strategy reflects continued focus on further growth in this sector. We intend to grow our income from fee based services in this sector by offering new products and services and by cross-selling our offerings to our customers. While we anticipate continued demand in this area, we cannot assure you that our retail portfolio will continue to grow at the high rates we have recently experienced. We face significant challenges in our new businesses and our international expansion plans. We intend to diversify our products and services, particularly in retail banking . For example, we have recently begun selling credit cards and non-life insurance (i.e., property and casualty) policies. Although we are currently not selling our own products in these areas, we may do so in the future. We also sell mutual fund products through a joint venture. Selling our own products will require us, our subsidiaries or our joint ventures to take balance sheet risk in these areas. The new products that we may develop and sell may not be profitable in the initial years of operation and are subject to start-up risks and as well as general risks and costs associated with the respective businesses. In addition, we have recently opened representative offices in London ( United Kingdom ), Almaty ( Kazakhstan ) and Shanghai ( China ) and a branch in Kabul ( Afghanistan ). We may also expand into other countries. The skills required for our international business could be different from those required for our domestic operations. There is a high level of competition in these new businesses, which may impede our growth in these businesses. xiii We face challenges in restructuring and rationalizing our branch network and improving the productivity rates of our employees. We have an extensive network of over 4,000 branches, many of which are in rural and semi-urban areas. Several of our older “legacy” branches are undergoing renovation to modernize them and upgrade their technology, which will involve substantial capital expenditure. Because we have these legacy branches, the productivity rate of our employees is lower than many of our private competitors which have newer infrastructure and which initially computerized their branches earlier than us. We are also in the process of rationalizing our branch network which will involve closing or merging some branches and opening others, which we may not be able to fully accomplish within the expected time frames or at the expected cost. We have high concentrations of loans to certain customers and to certain sectors and if a substantial portion of these loans were to become non-performing, the quality of our loan portfolio could be adversely affected. As of September 30, 2004, our total outstanding exposure was Rs. 1,129.2 billion. Our total outstanding exposure includes both principal outstanding, interest and other dues on advances and investments (collectively, our fund based exposure) and the outstanding amount of guarantees, letters of credit and other non-fund based exposures (collectively, our non-fund based exposures). Our total exposure (including our funded exposure and 100% of our non-fund based limit or outstandings, whichever is higher) to our ten largest borrowers, which include single borrowers and borrower groups, in the aggregate accounted for approximately 9.6% of our total outstanding exposure as of September 30, 2004. Our exposure to our largest single borrower as of September 30, 2004 accounted for approximately 1.0% of our total outstanding exposure and 17.8% of our capital funds (comprising Tier 1 and Tier 2 capital, as defined in the Indian banking regulations). Our exposure to our largest borrower group as of September 30, 2004 accounted for approximately 1.6% of our total outstanding exposure and 28.6% of our capital funds. Credit losses on these large single borrower and group exposures could adversely affect our business and financial condition. Our internal policies limit our credit exposure to any particular industry to 10.0% of our total credit. We determine our industry-wise exposures by aggregating our fund based exposures in respect of accounts of Rs. 10 million or larger within each industry. See the section titled “Business - Risk Management - Credit Risk - Prudential Limits” on page 67. As of September 30, 2004, our five largest industry exposures other than priority sector exposures comprised infrastructure, state electricity boards, iron and steel, financial services and housing (including both direct and indirect lending to corporate borrowers, housing development authorities and state governments) and which in the aggregate constituted 40.6% of our total fund based exposures in respect of accounts larger than Rs. 10 million. Our aggregate funded exposure to the top five borrowers in these industries together represented 55.7% of our aggregate funded credit exposure to these industries. Financial difficulties in these industries could adversely affect our business and financial condition. As of September 30, 2004, 16.3% of our advances were to borrowers that we rate as being in the “marginally acceptable grade” and 14.9% of our advances were to borrowers that we rate as “sub-investment grade” under xiv our internal rating system. Our sub-investment grade borrowers, in particular, could be especially vulnerable if economic conditions should worsen or economic growth rates were to slow, which could adversely affect our business and financial condition. We may experience delays in enforcing our collateral when borrowers default on their obligations to us, which may result in failure to recover the expected value of the collateral. Although we typically lend on a cash-flow basis, we take collateral for a large proportion of our loans, consisting of liens on inventory, receivables and other current assets, and in some cases, charges on fixed assets and financial assets, such as marketable securities. As of September 30, 2004, over 86% of our advances were secured by tangible assets, such as real assets (including property, plant and equipment, liens on inventory, receivables and other current assets), fixed assets, the assets being financed or a charge or mortgage of agricultural land or immovable property. In India, foreclosure on collateral generally requires a petition to a court or tribunal. An application, when made, may be subject to delays and administrative requirements that may result, or be accompanied by, a decrease in the value of the collateral. Delays can last for several years, leading to deterioration in the physical condition and market value of the collateral. Although the constitutional validity of the recent Securitisation and Reconstruction of Financial Assets and Enforcement of Security Interest Act, 2002 (“Securitisation Act”) which is intended to strengthen the rights of lenders, has been upheld by the Supreme Court of India, the precise mechanisms for applying the Act are still evolving. As a result of the foregoing factors, as well as other factors such as defects in the perfection of collateral, may not be able to realize the full value of our collateral. Our funding is primarily through short-term deposits, and if depositors do not roll over deposited funds on maturity or if we are unable to continue to increase our deposits, our business could be adversely affected. Most of our funding requirements are met through short-term and medium-term funding sources, primarily in the form of deposits. As of September 30, 2004, 85.9% of our funding consisted of deposits and 38.9% of our funding consisted of demand deposits and savings deposits. A portion of our assets have long term maturities, creating a potential for funding mismatches. In our experience, a substantial portion of our customer deposits has been rolled over upon maturity and has been, over time, a stable source of funding. However, in the event that a substantial number of our depositors do not roll over deposited funds upon maturity, our liquidity position and business could be adversely affected. The average cost of our deposits was 4.6% for the first six months of fiscal 2005. If we are unable to maintain or increase our base of low cost deposits, our overall cost of funds could increase which could have an adverse effect on our business and our ability to grow. The Government will continue to hold a majority interest in us following this Issue and will therefore be able to significantly affect the outcome of shareholder voting. After the completion of this Issue and the Capital Reduction, the Government will own at least 57.80% of our outstanding Equity Shares and will be able to appoint seven directors of a total of thirteen directors. Consequently, the Government will continue to have a controlling interest in us and will xv also be able to determine a majority of our board of directors. Further, the Bank Acquisition Act limits the voting power of our shareholders by requiring that none of our shareholders other than the Government shall be entitled to exercise voting rights in respect of shares held by them in excess of 1% of the total voting rights of all of our shareholders. Therefore, the outcome of most proposals for corporate action requiring the approval of our Board of Directors or shareholders will be largely controlled by the Government. Furthermore, given the importance of the banking industry to the Indian economy, the Government, through policy directives, could require us to take actions and enter into transactions such as the acquisition of other banks or financial institutions that are in financial difficulty in order to serve the public interest of India and not necessarily to maximize our profits. We may undertake mergers or acquisitions which may pose management and integration challenges. We have in the past engaged in acquisitions and strategic partnerships. In February 2003, Nedungadi Bank was merged with us. While we believe that we have since successfully integrated the operations of this bank with our own, the merger increased our gross NPA portfolio by approximately Rs. 5.2 billion. Our gross NPA portfolio as of March 31, 2003 was Rs. 49.8 billion. We may make further acquisitions and investments, which may not necessarily contribute to our profitability and may require us to assume high levels of debt or contingent liabilities, as part of such transactions. We could experience difficulty in combining operations and cultures and may not realize the anticipated synergies or efficiencies from such transactions. If IFCI Limited (“IFCI”) is merged with us without our conditions to the merger being accepted, our business and financial condition could be adversely affected. IFCI was set up by the Government to provide financial support to the industrial sector in India. As of March 31, 2004, IFCI had Rs. 159.2 billion in assets, Rs. 11.0 billion in total income, Rs. 32.3 billion in loss after provisions and extraordinary items and 119.6 billion gross non performing assets. The Government requested that we examine a proposal to merge IFCI with our bank and our board of directors agreed in principle to consider the proposal, subject to several conditions, which we conveyed to the Government. These conditions include the completion of due diligence, the acquisition of only the performing assets of IFCI, and several other conditions designed to ensure that the merger does not have an adverse financial or operational impact on us. If IFCI is merged with us without our conditions being accepted, our business and financial condition would be adversely affected. It has been reported in the Indian press that the Government is considering various alternatives regarding IFCI, including merging IFCI with the Industrial Development Bank of India. No final decision in this regard has been conveyed to us. Problems in extending the roll-out of our Core Banking Solution and upgrading our technology could adversely affect our ability to expand our products and services across our branch network. As of September 30, 2004, our Core Banking Solution (“CBS”) was implemented in 694 branches. We are planning to increase its coverage to between 1,500 and 2,000 branches by March 31, 2006. xvi Our CBS provides interconnectivity among our branches and is required for us to provide many of the products and services we have introduced to target customers, such as internet banking and our deposit accounts having the “sweep in, sweep out” feature. If we are unable to successfully extend the roll-out of the CBS across our branch network, it will be difficult for us to expand our products and services across our branch network. In addition, we have only recently computerized the operations of all of our branches and we may experience difficulties in adapting to these computerized technologies. Significant security breaches in our computer systems and network infrastructure and fraud could adversely impact our business. We seek to protect our computer systems and network infrastructure from physical break-ins as well as security breaches and other disruptive problems caused by our increased use of the internet. Computer break-ins and power disruptions could affect the security of information stored in and transmitted through these computer systems and networks. These concerns will intensify with our increased dependence on technology. We employ security systems, including firewalls and password encryption, designed to minimize the risk of security breaches. To mitigate these risks, we intend to continue to implement adequate security technologies and establish appropriate operational procedures. However, these may not be sufficient to prevent fraud, break-ins, damage and failures. A significant failure in security measures could have an adverse effect on our business. System failures and calamities could adversely impact our business. Given the increasing share of retail products and services and transaction banking services to our business, the importance of systems technology to our business has increased significantly. Our principal delivery channels include our branches and ATMs. We have a centralized backup system for our interconnected branches and the back-up systems for our other branches are based locally. Any failure in our systems, particularly those utilized for our retail products and services and transaction banking, or the occurrence of calamities such as earthquakes, tsunamis and cyclones that affect areas in which we have a significant presence, could significantly affect our operations and the quality of our customer service. The provisions we have made in our financial statements on account of the impending banking industry wage settlement are estimates based on past experience and our actual expenditure following the wage settlement may vary from the amounts provided for. There has been no wage settlement in the banking industry since 1997, although one was due in 2002. The Indian Banking Association recently reached a broad understanding with banking industry employees that the maximum wage increase will be 13.25%. As described in the section titled “Management’s Discussion and Analysis of Financial Condition and Results of Operations” on page 90 and “Recent Developments” on page 117, we have made provisions in our financial statements on account of the wage settlement for the period after October 31, 2002, which is when the previous wage settlement expired. The understanding reached with the banking industry employees is not xvii final or binding, and the amount of our provisions is an estimate based on our past experience. The actual expenditure we will have to incur following the wage settlement may vary from the amounts we have provided for. In order to sustain our growth, we will need to maintain a minimum capital adequacy ratio. There is no assurance that we will be able to access the capital markets when necessary to do so. The RBI requires a minimum capital adequacy ratio of 9% to our total risk weighted assets. We must maintain this minimum capital adequacy level to support our continuous growth. The implementation of the Basel II capital adequacy standards could also result in a decline in our capital adequacy ratio. Our capital adequacy ratio as of September 30, 2004 on a historical, unrestated basis was 12.58%. If the Basel II standardized method were applied on a pro forma basis as of September 30, 2004, we would expect our capital adequacy ratio as of that date to be 9.85%. Our ability to support and grow our business could be limited by a declining capital adequacy ratio if we are unable to or have difficulty accessing the capital markets. Our contingent liabilities could adversely affect our financial condition. As of September 30, 2004, we had contingent liabilities of Rs. 379.5 billion. These included liabilities on account of forward exchange contracts of Rs. 224.1 billion, guarantees of Rs. 59.4 billion and acceptances, endorsements and other obligations of Rs. 90.9 billion. If these contingent liabilities fully materialize, our financial condition could be adversely affected. Our subsidiary PNB Gilts Ltd. incurred a loss in the first half of fiscal 2005. Further, our subsidiary PNB Asset Management Company (PNB AMC) Limited has negative reserves and surplus. Our subsidiary PNB Gilts Ltd., which had profits of Rs. 1.13 billion, 0.93 billion and 1.07 billion in fiscal 2002, 2003 and 2004, and incurred a loss of Rs. 0.87 billion in the first six months of fiscal 2005. The main reason for the loss was trading losses incurred by PNB Gilts as a result of increasing interest rates during the period. If these losses were to continue, it could adversely affect our financial performance. As of March 31, 2004, our subsidiary PNB AMC had negative reserves and surplus of Rs. 71.65 million on account of accumulated losses. The schemes managed by PNB AMC have been merged with the schemes of Principal Mutual Fund. The Board of Directors of our Bank in its meeting held on March 29, 2003 accorded in-principle approval for the merger of PNB AMC with us. For further details see the section titled “Our Promoter, Subsidiaries and Group Companies” on page 127. Our subsidiary PNB Gilts Ltd. did not meet the promises made in its initial public offering. Our subsidiary PNB Gilts Ltd. conducted its initial public offering in July 2000. The prospectus for the offering contained financial projections for the financial year ended March 31, 2001 and the same has not been met by the company. The company had projected a profit before tax of Rs. 1399.1 million for fiscal 2001. However the actual profit before tax less than the projected amount by Rs. 592.2 million. For more details, please refer to the section titled “Our Promoter, Subsidiaries and Group Companies – PNB Gilts Limited—Promise v/s Performance” on page 128. xviii We are involved in a number of legal and regulatory proceedings that, if determined against us, could have a material adverse impact on us. There are two criminal cases against us and another criminal complaint against Mr. S.S. Kohli, our Chairman and Managing Director. There are two cases against our Bank relating to alleged violations of foreign exchange regulations in India and the total liability of our Bank amounts to Rs 3.5 million. There are 79 cases filed against us for disputes in relation to forged signatures, non-tallying of signatures, breach of trust and fraudulent withdrawal of money by allegedly unauthorized persons (including our employees) in which the total amount claimed against us amounts to Rs. 313.85 million. There are 104 cases filed against us relating to disputes on account of alleged non-deposit, non- realization or loss of cheques and alleged wrongful refusal to sanction certain facilities and overdrafts by us or release of sums to certain persons or wrongful debits in a customer’s account or refusal to honor bank guarantees, letters of credits, bills of lading and other such instruments including fixed deposit receipts maintained with us. The total amount claimed against us in these cases is Rs. 19,331.15 million. There are 211 consumer cases filed against us and the total amount claimed in such cases is Rs. 78.85 million. There are six cases pending against us wherein disputes have arisen in relation to the interest charged by us. The total amount claimed in these cases is Rs. 7.49 million. A number of customers have filed counter claims and counter suits against recovery suits or other actions undertaken by us. There are 24 such cases pending against us and the total amount in dispute is Rs. 230.70 million. 17 cases have been filed against us in relation to arrears or enhancement of the rent of the premises presently or formerly occupied by us. The total amount claimed in such cases is Rs. 53.5 million. There are 12 cases where our bank has been impleaded as a third party and the total amount claimed against us in such cases is Rs. 28.67 million. There are 13 cases against some branches of our Bank in which irregularities with regard to the public issues of certain other companies in which our Bank was acting as a intermediary and in which action has been taken by regulators against the said branches of the Bank. There are 701 labour related cases pending against us and the total amount claimed by the plaintiffs in these cases is approximately Rs. 161.77 million. There are 32 other cases pending against us and the total amount claimed in such cases is Rs. 26.05 million. For details of these legal proceedings, please see the section titled “Outstanding Litigation and Material Developments” on page 185. We are involved in a number of income tax and sales tax cases which if determined against us could have a material adverse impact on us. There are disputes relating to income tax assessments for the assessment years 1992-1993, 1994- xix 1995 to 2003-2004 in which the aggregate amount claimed against us was Rs. 485.94 million as of December 31, 2004. We have already paid the entire amount claimed by the income tax department under protest except Rs. 71 million in relation to assessment year 1997-98 on account of disallowance of software and bond issue expenditure due to rectification application filed and pending with the IT Department. There are three sales tax cases against us in which the liability imposed on us amounts to Rs. 6.67 million. However, a sum of approximately Rs. 2 million has already been paid as per order of the tax department and therefore, the aforesaid demand is reduced to Rs. 4.58 million. Our branches have a number of regulatory cases against them. There are 13 notices against some branches of our Bank in which irregularities with regard to the public issues of certain companies in which our Bank was acting as a intermediary and in which action has been taken by regulators against these branches. These actions include recommendations from SEBI to suspend the registration of some branches and debarring the concerned branches from accepting any merchant banking business. If these cases are adversely determined, or the sanctions continue, they could have an adverse impact on the business of these branches. Our subsidiaries, joint ventures and associate companies are involved in a number of legal proceedings, which if determined against them could have an adverse impact on their operations. Our subsidiaries, joint ventures and associate companies are involved in a number of legal proceedings including income tax cases, regulatory cases, criminal cases, consumer cases and service matters, which if determined against them could have a material adverse impact on their operations. The aggregate amount claimed against our associates is approximately Rs. 138.54 million, out of which amounts of Rs. 81.93 million is already deposited, the contingent liabilities of our associates that are not provided for amount to Rs. 5.33 million, the contingent liabilities of our joint ventures that are not provided for amount to Rs. 1.92 million, the aggregate amount claimed against our subsidiaries is approximately Rs. 56.06 million and the contingent liabilities of our subsidiaries that are not provided for amount to Rs. 31.07 million. Our financial statements for the six months ended September 30, 2003 and 2004 have not been audited. In terms of the guidelines issued by the RBI applicable to public sector banks and the Listing Agreement, the financial statements of the Bank for the six months ended September 30, 2003 and 2004 have not been audited. However, they have been subjected to a limited review by our Auditors. The related party disclosures in this document do not include transactions with subsidiaries and the regional rural banks in which we have an interest. As per RBI circular no. DBOD.No.BP. BC. 89 /21.04.018/2002-03 dated March 29, 2003 on Guidelines on the Compliance of Accounting Standards by Banks, all nationalized banks (like our Bank) are exempt from disclosing the transactions with their subsidiaries as well as the RRBs sponsored by xx them. Hence no such disclosure has been made in this Red Herring Prospectus. Further, we are not disclosing transactions with associates in the audited accounts. We are yet to receive certain statutory approvals required in the ordinary course of our business. We have applied to the RBI for a one year extension of a license bearing the number BL.LK. 98/2003 relating to an offshore banking unit in the Noida special economic zone. We have applied to the Governor of the Central Bank of the United Arab Emirates requesting permission to establish a representative office in Dubai. We have applied to RBI for setting up a joint venture with Principal Financial Group of the United States of America for a life insurance and pension company and an insurance brokerage company. Some of our immovable properties have certain irregularities in title. Some of our immovable properties for our branches, offices and residences which are either owned by us or occupied by us have one or more of the following irregularities in title: ● the conveyance deeds for transfer of property have not been executed; ● the agreements to sell or conveyance deeds have not been registered in the land records maintained by the concerned Sub-Registrar of Assurances; or ● lease deeds have not been executed or have expired and have not been renewed. For details of disputes relating to our properties, see the section titled “Outstanding Litigation and Material Developments” on page 185. Your holdings may be diluted by additional issuances of equity and any dilution may adversely affect the market price of our Equity Shares. There is a risk that we may be required to finance our growth through additional equity offerings. Any future issuance of our Equity Shares, or sales of a large number of our Equity Shares by major shareholders, could dilute the positions of investors in our Equity Shares and could adversely affect the market price of our Equity Shares. You will not receive the Equity Shares you purchase in this Issue until several days after you pay for them, which will subject you to market risk. The Equity Shares you purchase in this Issue will not be credited to your demat account with depositary participants until approximately fifteen days from the Bid Closing Date/Issue Closing Date. You can start trading your Equity Shares only after they have been credited to your demat account. Since the Equity Shares are already traded on the Stock Exchanges, you will be subject to market risk from the date you pay for the Equity Shares to the date they are credited to your demat account. Further, there can be no assurance that the Equity Shares allocated to you will be credited to your demat account, or that trading in the Equity Shares will commence, within the time period specified above. xxi However, as stated on page 11, we will pay interest at 15% per annum if there is any delay for crediting the demat shares beyond the above-stated period. This risk factor is for the information of investors and it does not in any way dilute the right of investors and our obligations. External Risk Factors The Indian and global banking industry is very competitive and the ability of banks to grow depends on their ability to compete effectively. There is competition among public and private sector Indian commercial banks as well as foreign commercial banks in India. Some of these banks are smaller and may be more flexible and better positioned to take advantage of market opportunities than public sector banks. In particular, private banks may have operational advantages in implementing new technologies, rationalizing branches and recruiting employees through incentive-based compensation. Additionally, both the Indian and global financial sector may experience further consolidation, resulting in fewer banks and financial institutions. It has been recently reported in the press that the Government may soon permit foreign banks to establish wholly-owned subsidiaries in India and invest up to 74% in Indian private sector banks and eliminate or modify voting restrictions on shareholders of banks. These measures, if implemented, are likely to further increase competition and may stimulate consolidation in the Indian banking industry. In addition, the Government is actively encouraging banks and other financial institutions to significantly increase their lending to the agriculture sector, which will make this segment more competitive. These competitive pressures affect the Indian banking industry as a whole, including our Bank, and our growth will depend in large part on our ability to respond in an effective and timely manner to these competitive pressures. Banking is a heavily regulated industry and material changes in the regulations which govern us could adversely affect our business. Banks in India are subject to detailed supervision and regulation by the RBI. In addition, banks are subject generally to changes in Indian law, as well as to changes in regulation and government policies and accounting principles. It has recently been indicated in the press that the laws and regulations governing the banking sector could change in the near future and any such changes could adversely affect our business, our future financial performance, our shareholders’ funds and the price of our equity shares, by requiring a restructuring of our activities, increasing costs or otherwise. For information on the regulations and policies applicable to our Bank see section titled “Regulations and Policies” on page 151. There are a number of restrictions under the Bank Acquisition Act, the Bank Regulations, the Bank Scheme and various RBI notifications, press notes and circulars which affect our operating flexibility and affect or restrict the rights of investors. These restrictions are different from those normally applying to shareholders of companies incorporated under the Companies Act, and include the following: xxii ● We can increase our paid up capital only with the consent of the Government and in consultation with the RBI in such manner as may be prescribed and the shareholding of the Government shall not go below 51% of our paid up capital. ● The Government has control over policy matters relating to us and the RBI has the power to caution or prohibit us from entering into any particular transaction or class of transactions. ● Foreign investment is subject to an overall statutory limit of 20% of our paid up capital. The RBI has fixed a cut off point at 18% for the purposes of effective monitoring. Once the aggregate net purchases of equity shares of the respective bank by FIIs/NRIs/PIOs reaches the cut off point, which is 2% below the overall limit of 20%, the RBI cautions all designated bank branches so as not to purchase any more equity shares in the respective bank on behalf of NRIs/PIOs without prior approval of the RBI. For details see the section titled “Regulations and Policies-Restriction on Transfer of Shares” on page 176. ● No shareholder, other than the Government, is entitled to exercise voting rights in respect of any shares held by it in excess of one per cent of the total voting rights of all our shareholders. ● There are restrictions on payment of dividend and on rights relating to unclaimed dividends. For information on these restrictions see section titled “Regulations and Policies - Restrictions on Payment of Dividends” on page 177. ● Certain restrictions on opening a new place of business and transferring an existing place of business, require the approval of RBI (except in certain circumstances) which may hamper the operational flexibility of the Bank. For information on these restrictions see section titled “Regulations and Policies – Regulations relating to the Opening of Branches” on page 168. ● We have to maintain assets in India equivalent to not less than 75% of our demand and time liabilities in India, which in turn may restrict us from building overseas asset portfolios and exploiting overseas business opportunities. ● There are no provisions for requiring us to send compulsory statutory reports to our shareholders prior to a general meeting of the shareholders and our shareholders do not have the right to approve our accounts, the report of our Board of Directors on our activities for the period covered by the accounts and the auditors’ report on our accounts. However, we are required by our listing agreements with the Indian stock exchanges to send annual reports to our shareholders prior to our annual general meeting. ● Rights of minority shareholders statutorily available in the case of a company incorporated under the Companies Act are not available to our shareholders. For a comparison of the rights available to shareholders of a company incorporated under the Companies Act with the rights statutorily available to our shareholders, see the section titled “Regulations and Policies - Comparative Table of Rights of Shareholders under Companies Act, 1956 and under Regulations Applicable to Corresponding New Banks” on page 151. xxiii We are exposed to certain risks of the Indian financial system and could be impacted by the financial difficulties of other financial institutions in India. As an Indian bank , we are exposed to the risks of the Indian financial system which in turn may be affected by financial difficulties and other problems faced by Indian financial institutions. As an emerging market system, the Indian financial system faces risks of a nature and extent not typically faced in developed countries. Additionally, the credit risk of our borrowers is higher than in more developed countries. Unlike several developed countries, India’s nationwide credit bureau is still at a nascent stage, which may affect the quality of information available to us about the credit history of our borrowers, especially individuals and small businesses. Certain Indian financial institutions have experienced difficulties during recent years. Some co- operative banks have also faced serious financial and liquidity crises. The problems faced by individual Indian financial institutions and any instability in or difficulties faced by the Indian financial system generally could create adverse market perception about Indian financial institutions and banks. This in turn could adversely affect our business and our future financial performance. Exchange rate fluctuations may have an impact on our financial performance. As a financial organization we are exposed to exchange rate risk. Adverse movements in foreign exchange rates may impact our business, our future financial performance, our shareholders’ funds and the price of our Equity Shares. A slowdown in economic growth in India could cause our business to suffer. The Indian economy has shown sustained growth over the last few years with GDP growing at 8.5% in fiscal 2004, 4.0% in fiscal 2003 and 5.8% in fiscal 2002. During the first six months of fiscal 2005, GDP grew at 7.0% compared to 6.9% during the first six months of fiscal 2004. However, growth in industrial and agricultural production in India has been variable. Industrial growth was 6.6% in fiscal 2004 compared to 6.2% in fiscal 2003 and 3.5% in fiscal 2002. Industrial growth was 7.9% during the first six months of fiscal 2005. Agricultural production grew by 9.6% in fiscal 2004 compared to a 5.2% decline in fiscal 2003 and growth of 6.5% in fiscal 2002. Agricultural production grew by 1.5% in the first six months of fiscal 2005. In its mid-term review of annual policy published on October 26, 2004, the RBI reduced its GDP growth forecast for fiscal 2005 from 6.5-7% to 6.0-6.5% as a result of a decline in agricultural growth because of insufficient rainfall, and increased its inflation rate forecast from 5.0% to 6.5%. Any slowdown in the Indian economy or volatility in global commodity prices, in particular oil and steel prices, could adversely affect our borrowers and contractual counterparties. With the importance of retail loans to our business, any slowdown in the growth of sectors like housing and automobiles could adversely impact our performance. Further, given the importance of the agricultural sector to our business, any slowdown in the growth of the agricultural sector could also adversely impact our performance. xxiv A significant change in the Government of India’s economic liberalization and deregulation policies could disrupt our business and cause the price of our Equity Shares to decline. Our assets and customers are predominantly located in India. The Government of India has traditionally exercised and continues to exercise a dominant influence over many aspects of the economy. Its economic policies have had and could continue to have a significant effect on private sector entities, including us, and on market conditions and prices of Indian securities, including our Equity Shares. The present Government, which was formed after the Indian parliamentary elections in April-May 2004, is headed by the Indian National Congress and is a coalition of several political parties. Any significant change in the Government’s policies or any political instability in India could adversely affect business and economic conditions in India and could also adversely affect our business, our future financial performance and the price of our Equity Shares. Natural calamities could have a negative impact on the Indian economy and cause our business to suffer. India has experienced natural calamities such as earthquakes, a tsunami, floods and drought in the past few years. The extent and severity of these natural disasters determines their impact on the Indian economy. For example, as a result of drought conditions in the country during fiscal 2003, the agricultural sector recorded a negative growth of 5.2%. The erratic progress of the monsoon in 2004 has also adversely affected sowing operations for certain crops. Further prolonged spells of below normal rainfall or other natural calamities could have a negative impact on the Indian economy, adversely affecting our business and the price of our Equity Shares. Based on reports to date, the tsunami that struck the eastern coast of India and other Asian countries on December 26, 2004 did not result in any fatalities or serious injuries to our staff nor any damage to our properties, and we do not believe we had significant aggregate loan or other exposure to persons directly affected by the disaster. However, the full effects of the tsunami, particularly its effects on economic conditions in India generally, are not yet known and therefore we cannot predict at this time whether it will have a significant effect on our business. Terrorist attacks, civil unrest and other acts of violence or war involving India and other countries could adversely affect the financial markets and our business. Terrorist attacks, such as the ones that occurred in New York and Washington, D.C. on September 11, 2001, New Delhi on December 13, 2001, Gandhinagar in Gujarat on September 24, 2002, Bali on October 12, 2002 and Mumbai on August 25, 2003 and other acts of violence or war may negatively affect the Indian markets where our Equity Shares trade and also adversely affect the worldwide financial markets. These acts may also result in a loss of business confidence, make travel and other services more difficult and ultimately adversely affect our business. After the December 13, 2001 attack in New Delhi and a terrorist attack on May 14, 2002 in Jammu, India, diplomatic relations between India and Pakistan became strained and there was a risk of intensified tensions between the two countries. The governments of India and Pakistan have recently xxv been engaged in conciliatory efforts. However, any deterioration in relations between Indian and Pakistan might result in investor concern about stability in the region, which could adversely affect the price of our Equity Shares. India has also witnessed civil disturbances in recent years and it is possible that future civil unrest as well as other adverse social, economic and political events in India could have an adverse impact on us. Regional or international hostilities, terrorist attacks or other acts of violence of war could have a significant adverse impact on international or Indian financial markets or economic conditions or on Government policy. Such incidents could also create a greater perception that investment in Indian companies involves a higher degree of risk and could have an adverse impact on our business and the price of our Equity Shares. Any downgrading of India’s debt rating by an international rating agency could have a negative impact on our business. Any adverse revisions to India’s credit ratings for domestic and international debt by international rating agencies may adversely impact our ability to raise additional financing, and the interest rates and other commercial terms at which such additional financing is available. This could have a material adverse effect on our business and future financial performance our ability to obtain financing for capital expenditures, and the price of our Equity Shares. Notes: ● The RBI conducts regular inspections of banking companies under the provisions of the Banking Regulation Act. The reports of the RBI are strictly confidential. We are in discussions with the RBI in respect of observations made by the RBI in their reports for prior periods. The RBI does not permit disclosure of its inspection report and all disclosures in this Red Herring Prospectus are on the basis of our management and audit reports. ● Public issue of up to 80,000,000 Equity Shares for cash at a price of Rs [●] per Equity Share aggregating Rs. [●] million. The Issue comprises a Net Issue to the public of 64,000,000 Equity Shares, a reservation, i.e., Employees Reservation Portion of up to 8,000,000 Equity Shares and a reservation, i.e., Existing Shareholders Portion of up to 8,000,000 Equity Shares. ● The net worth of our Bank before the Issue (based on our restated financial statements as of September 30, 2004) was Rs. 50.96 billion (on an unconsolidated basis). ● The average cost of acquisition of Equity Shares by the President of India, acting through the MoF, our Promoter, is Rs. 10 per Equity Share and the book value per Equity Share based on our restated financial statements as of March 31, 2002 was Rs. 130.4, as of March 31, 2003 was Rs. 132.6, as of March 31, 2004 was Rs. 165.2 and as of September 30, 2004 was Rs. 192.10 per Equity Share. xxvi ● Refer to the notes to our financial statements relating to related party transactions in the section titled “Related Party Transactions” on page 149. Our aggregate related party transactions with key managerial personnel are as follows: Item Amount Amount Amount (Rs. in million) (Rs. in million) (Rs. in million) (2001-02) (2002-03) (2003-04) Remuneration 0.9 1.0 1.1 ● Further the related party transactions with associates (unaudited) are given below: Assets Care Enterprises Ltd. Rs. in million FY 2002 FY 2003 FY 2004 Share Capital held by Bank - - 13 Principal PNB Asset Management Company Private Limited Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Share Capital held by Bank - - 30 Principal Trustee Company Private Limited Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Share Capital held by Bank - - 0.30 xxvii PNB Principal Financial Planners Private Limited A sum of Rs. 15,000,000 was paid to this company on October 14, 2004 by our Head Office, Treasury Division, towards the Bank’s share of capital contribution. Further, the company has paid a sum of Rs. 0.9 million royalty for use of the Bank’s name. Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Capital Contribution to joint venture - - 15 Royalty to the Bank - - 0.9 Everest Bank Limited Rs. in million FY 2002 FY 2003 FY 2004 Share Capital held by Bank 33.0 39.6 39.6 Dividend received by Bank - 7.9 7.9 Technical Services fee received by Bank 2 2 2 ● None of our directors has, either directly or indirectly, undertaken transactions in our Equity Shares in the six months preceding the date of this Red Herring Prospectus. ● Investors may contact the BRLMs for any complaints, information or clarifications pertaining to the Issue. ● Investors are advised to refer to the section titled “Basis for Issue Price” on page 241. Investors should note that in case of over-subscription in the Issue, allotment will be made on a proportionate basis to Retail Individual Bidders, Employees, Existing Shareholders and Non Institutional Bidders. Please refer to the section titled “Statutory and Other Information - Basis of Allocation” on page 244. xxviii SUMMARY You should read the following summary together with the section titled “Risk Factors” on page x, our financial statements and the more detailed information about us included elsewhere in this Red Herring Prospectus. Overview We are a leading public sector commercial bank in India, offering banking products and services to corporate and commercial, retail and agricultural customers. We started our operations in 1895 and since then have grown to become India’s third largest bank in terms of assets and second largest bank in terms of number of branches. Although we began our operations in the agriculturally rich areas of northern India, we have expanded our operations to provide products and services to over 35 million customers across India through more than 4,000 branches. Our banking operations for corporate and commercial customers include a range of products and services for large corporate customers as well as for small and middle market businesses and government entities. We cater to the financing needs of the agricultural sector and have created innovative financing products for farmers. We also provide significant financing to other priority sectors including small scale industries. We offer a wide range of retail credit products including home loans, personal loans and automobile loans. Through our subsidiaries and joint ventures, we deal in Indian government securities and provide housing finance and asset management services. Through our treasury operations, we manage our balance sheet, including the maintenance of required regulatory reserves, and seek to maximize profits from our trading portfolio by taking advantage of market opportunities. Since 1969, when we became a public sector bank, we have managed to continue to grow our business while maintaining a strong balance sheet. As of September 30, 2004, our total deposits represented 85.9% of our total liabilities. On average, interest free demand deposits and low interest savings deposits represented 43.8% of these deposits in the first six months of fiscal 2005. These low-cost deposits led to an average cost of funds excluding equity for the first six months of fiscal 2005 of 4.7%. As of September 30, 2004, our gross and net non-performing assets constituted 7.65% and 0.30% of our gross and net advances, respectively. In fiscal 2004 our total income was Rs. 96.5 billion and our net profit was Rs. 11.1 billion before adjustment and Rs. 10.6 billion after adjustment as part of the restatement of our financial statements for this Issue. In the first six months of fiscal 2005 our total income was Rs. 51.9 billion and our net profit was Rs. 7.4 billion before adjustment and Rs. 7.9 billion after adjustment. Between fiscal 2002 and 2004, our total income grew at a compound annual rate of 12.5%, our unadjusted and adjusted net profit grew at a compound annual rate of 40.4% and 37.4%, respectively, and our total deposits and total advances grew at a compound annual growth rate of 17.1% and 17.2%, respectively. We intend to maintain our position as a cost efficient and customer friendly institution that provides comprehensive financial and related services. We seek to achieve this by continuing to adopt technology which will integrate our extensive branch network. We intend to grow by cross-selling various financial products and services to our customers and by expanding geographically in India and internationally. We are committed to excellence in serving the public and also maintaining high standards of corporate responsibility. In line with our philosophy of aiding India’s development we have opened branches in many rural areas. Strategy Our goal is to further strengthen our position as one of India’s premier commercial banks and to increase our profitability by providing a comprehensive range of products and services and superior customer service. Our business strategy emphasizes the following elements: Expand our business as the Indian economy grows by using our extensive domestic branch network to deliver high quality service that is tailored to the needs of our customers The Indian economy is currently experiencing a high rate of growth. The consequent increase in the size and incomes of the middle and upper-income classes has resulted in significant expansion in India’s banking and financial services industry. We believe that we can benefit from the growth of the Indian economy and increase our market share by continuing to emphasize high quality service through our extensive network of over 4,000 branches and our comprehensive product offerings which are tailored to our customers. To this end, we intend to continue to expand and at the same time restructure and rationalize our branch network to make our services more efficient. We also have introduced specialized branches that cater to the specific 1 needs of various categories of customers. We intend to continue to implement these measures so as to ensure that we have one of the largest, as well as one of the most efficient, delivery networks in India. Strengthen and expand our corporate and commercial franchise We seek to build on our historical strength in the corporate and commercial sector by expanding our product and service offerings to large corporate customers and government entities as well as growth-oriented small and medium sized businesses. We seek to increase our customer base by offering innovative products and servicing the needs of our corporate and commercial customers through specialized branches, including those targeted at large and medium sized corporate customers and those targeted at small scale industries. Additionally, we seek to cross-sell our fee-based products and services to our current customer base and thereby increase our income from these products and services. Expand retail opportunities by effectively leveraging our customer base and increasing our product offerings With the increase in household income levels in India and the consequent need for diversified financial services, the retail sector has emerged as a rapidly growing opportunity for banks with the skills and infrastructure to adequately service this market. The keys to our retail strategy are wide distribution, convenient customer service, a full range of products and prudent risk management. Cross-selling of a wide range of banking services and insurance, credit and investment products to our customers is a critical aspect of our retail strategy as we leverage our current customer base to expand into new product areas. Maintain and enhance our franchise in the agriculture sector We intend to maintain and enhance our position as one of the leading banks for agricultural lending in India. In 2004, we exceeded the national goal for allocation of credit to the agricultural sector. We have introduced innovative products such as our Krishi credit cards, which provide farmers with convenient financing for their production, investment and consumption activities. We have a nationwide franchise in the agriculture sector, with concentration in northern India, one of the country’s most fertile areas. We intend to further expand our agriculture lending activities in other areas in India, including those which have historically not been adequately served. We also intend to expand our agriculture lending activities into areas such as horticulture and herbal cultivation. Another aspect of our strategy is to further strengthen our ties with the agricultural community by providing training and social support programs for the rural populace. Develop our technological capabilities to interconnect our branch network and improve our services We believe that technology is a critical differentiator in the banking industry and over the past few years have devoted substantial resources to upgrading our technology. We have electronically interconnected over 690 of our branches (as of September 30, 2004), which we believe is significantly more than any other bank in India. We intend to extend this to between 1,500 and 2,000 branches by March 31, 2006. We also intend to expand the delivery channels of our services and have introduced internet banking and set up over 450 ATMs. We aim to improve service by further using interconnectivity to implement centralized credit management, helpdesk services and data backup capabilities. Maintain high asset quality standards through comprehensive risk management We have maintained high quality loan and investment portfolios by carefully targeting our customer base and implementing a comprehensive risk assessment process and diligent risk monitoring and remediation procedures. We apply our credit risk rating process to advances in excess of Rs. 2 million. Additionally, we actively monitor our loans and reassess their credit ratings once a year or more frequently if they are at risk. We also apply aggressive remediation policies to recover non-performing loans. Our provisioning policies for non-performing assets are in accordance with the RBI guidelines. As of September 30, 2004 we had floating provisions of Rs. 13.9 billion for non-performing assets, which was significantly in excess of those required under the RBI guidelines. Our gross NPA coverage ratio was 96.4% as of September 30, 2004. Maintain our low cost of funds We have one of the lowest overall cost of funds in the Indian banking industry. We have achieved this through a large base of low cost deposits, with total deposits representing 90.7% of our funding as of September 30, 2004. Interest free demand deposits and low interest savings deposits constituted 10.3% and 34.9%, respectively, of our total deposits as of September 30, 2004. We believe we can maintain and enlarge this low-cost funding base by leveraging our extensive branch network and our customer base of over 35 million accounts and by offering a wide range of products and high quality customer service. 2 SUMMARY FINANCIAL INFORMATION The following table sets forth summary financial information derived from our unconsolidated financial statements. The financial information as of and for the fiscal years ended March 31, 2002, 2003 and 2004 and as of and for the six months ended September 30, 2003 and 2004 is based on our restated financial statements for those periods, which are included in this Red Herring Prospectus under the section titled “Financial Statements” on page F-1. The restated financial statements have been prepared in accordance with Indian GAAP and the SEBI Guidelines and have been restated as described in the auditors’ report attached thereto. The summary financial information presented below should be read in conjunction with the financial statements included in this Red Herring Prospectus, the notes thereto and the sections titled “Selected Statistical Information” on page72, “Management’s Discussion and Analysis of Financial Condition and Results of Operations” on page 90 and “Recent Developments” on page 117. Indian GAAP differs in certain significant respects from U.S. GAAP. For more information on these differences, see the section titled “Summary of Significant Differences between Indian GAAP and U.S. GAAP”, on page 270. SUMMARY PROFIT AND LOSS INFORMATION (In Rs. million) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 INCOME Interest Income Interest and discount on advances and bills 33,178.8 37,115.9 38,760.1 19,319.9 20,732.4 Income on investments 30,027.6 32,983.3 36,809.7 17,810.9 19,706.4 Other interest income 3,272.3 4,750.9 2,227.2 1,038.1 992.4 Total interest income 66,478.7 74,850.1 77,797.0 38,168.9 41,431.2 Other Income Commission, exchange and brokerage 4,339.9 4,800.1 5,519.0 2,498.2 3,218.0 Profit on sale of investments (net) 4,379.1 6,722.8 12,363.7 6,989.8 2,786.1 Others 1,058.2 980.2 786.1 1,002.3 4,499.2 Total other income 9,777.2 12,503.1 18,668.8 10,490.3 10,503.3 Total Income 76,255.9 87,353.2 96,465.8 48,659.2 51,934.5 EXPENDITURE Interest Expenditure Interest on deposits 41,216.2 41,625.6 39,264.4 19,744.6 21,006.4 Others 2,309.6 1,987.3 2,285.5 1,113.6 1,373.3 Total interest expenditure 43,525.8 43,612.9 41,549.9 20,858.2 22,379.7 3 (In Rs. million) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 Operating Expenses Payment to and provision for employees and wages 13,163.2 14,760.8 16,540.6 7,602.6 9,515.6 Other operating expenses 4,828.9 5,806.5 7,166.7 3,100.9 3,681.9 Total operating expenses 17,992.1 20,567.3 23,707.3 10,703.5 13,197.5 Total Expenditure 61,517.9 64,180.2 65,257.2 31,561.7 35,577.2 Gross profit before provisions and contingencies 14,738.0 23,173.0 31,208.6 17,097.5 16,357.3 Provisions and Contingencies Provision for non performing advances 3,913.3 4,729.7 4,010.4 2,405.2 1,182.1 Floating provision against NPAs* 2,400.0 3,600.0 7,930.0 5,730.0 — Provisions for income tax 1,981.5 3,406.6 6,599.9 2,803.5 2,831.2 Other provisions 819.3 3,014.7 1,581.4 643.6 4,991.8 Total provisions and contingencies 9,114.1 14,751.0 20,121.7 11,582.3 9,005.1 Net profit 5,623.9 8,422.0 11,086.9 5,515.2 7,352.2 ADJUSTMENTS ON ACCOUNT OF RESTATEMENT (In Rs. million) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 Adjustments for amortization of goodwill on account of amalgamation of Nedungadi Bank — (373.3) 373.3 186.7 — Adjustments for impact of wage revision — (460.0) (1,104.0) (552.0) 861.0 Tax impact of adjustments — 306.2 254.9 127.5 (303.9) Total adjustments — (527.1) (475.8) (237.8) 557.1 Transfer from other reserves — — — — 584.9 Adjusted net profit 5,623.9 7,894.9 10,611.1 5,277.4 8,494.2 * Floating provisions against NPAs has been explained in the section titled “Management’s Discussion and Analysis of Financial Condition and Result of Operations” on page 90. 4 SUMMARY BALANCE SHEET INFORMATION (In Rs. million) As of March 31, As of September 30, 2002 2003 2004 2004 ASSETS Investments 282,071.7 340,300.5 421,254.9 460,185.8 Advances 343,694.2 402,281.2 472,247.2 518,705.3 Balance with RBI 43,704.1 59,397.1 60,430.7 60,846.4 Other assets 56,295.3 56,876.3 66,735.3 57,945.4 Total Assets (A) 725,765.3 858,855.1 1,020,668.1 1,097,682.9 LIABILITIES Deposits 641,234.8 758,135.0 879,163.9 945,886.4 Borrowings 4,085.7 6,621.6 12,890.6 19,520.2 Other liabilities and provisions 38,220.7 41,662.9 59,130.2 55,033.6 Subordinated debt 11,798.6 15,928.6 23,578.6 23,428.6 Total liabilities (B) 695,339.8 822,348.1 974,763.3 1,043,868.8 NET ASSETS (C=A-B) 30,425.5 36,507.0 45,904.8 53,814.1 Share capital (D) 3,767.3 2,653.0 2,653.0 2,653.0 Reserves and surplus (E) 26,658.2 33,854.0 43,251.8 51,161.1 TOTAL (D+E) 30,425.5 36,507.0 45,904.8 53,814.1 5 THE ISSUE Issue 80,000,000 Equity Shares Of which: Reservation for Employees Up to 8,000,000 Equity Shares Reservation for Existing Shareholders Up to 8,000,000 Equity Shares Net Issue to the Public At least 64,000,000 Equity Shares Of which: Qualified Institutional Buyers Portion Up to 32,000,000 Equity Shares (allocation on a discretionary basis) Non-Institutional Portion At least 9,600,000 Equity Shares (allocation on a proportionate basis) Retail Portion At least 22,400,000 Equity Shares (allocation on a proportionate basis) Equity Shares outstanding prior to the Issue 265,302,500 Equity Shares Equity Shares outstanding after the Issue, 345,302,500 Equity Shares without giving effect to the Capital Reduction Equity Shares outstanding after the Issue, giving 315,302,500 Equity Shares effect to the Capital Reduction Objects of the Issue Please see the section titled “Objects of the Issue” on page 29. 6 GENERAL INFORMATION Authority for the Issue Our Board of Directors had authorised a fresh issue of up to 50,000,000 Equity Shares pursuant to a resolution passed at its meeting held on September 11, 2004. Our shareholders subsequently authorised the fresh issue of up to 50,000,000 Equity Shares, by a resolution passed unanimously at the general meeting of our Bank held on October 11, 2004 at New Delhi, subject to the approval of the Government, the RBI, SEBI and other applicable authorities. The shareholder resolution gave the Board the authority and power to accept any modification in the proposal as may be required or imposed by such authorities and as agreed to by the Board. Our Bank applied to the Government on October 14, 2004 for its sanction to a fresh issue of up to 50,000,000 Equity Shares. By its letter bearing number F.No.11/29/2004-BOA dated January 7, 2005, pursuant to Section 3 (2B) (c) of the Bank Acquisition Act, the Department of Economic Affairs, MoF, GoI conveyed its approval on the terms and conditions requiring an increased issue size of 80,000,000 Equity Shares and containing, as a condition to the approval, that the amount raised for 30,000,000 Equity Shares through the public offer should be returned to the Government at the Issue Price by March 31, 2005. Further, in terms of the Government Approval, the proportionate cost of the public issue to the extent of 30,000,000 Equity Shares is to be borne by the Government. Following the receipt of the Government Approval and in pursuance of the powers granted to the Board by the shareholders of our Bank in the extraordinary general meeting dated October 11, 2004, our Board, through a resolution dated January 13, 2005, authorized the Issue, subject to the conditions specified in the Government Approval. For further details see the sections titled “Risk Factors” on page x, “Capital Structure” on page 22 and “Government Approvals” on page 182. We have received permission from the Foreign Exchange Department, RBI vide their letter no.FE.CO.FID/4998/10.02.40(8435)/ 2004-05 dated February 2, 2005 permitting us to issue shares to NRIs / FIIs with repatriation benefits out of our public Issue of 80,000,000 Equity Shares. The RBI has imposed the condition that post-Issue, the non resident equity holding in our Bank should not exceed 20% of the paid up capital. The permission is further subject to the conditions laid down by the Government of India in their approval letter no. F.No.11/29/2004-BOA dated January 7, 2005, condition prescribed/stipulated by SEBI in this connection and the terms and conditions for issue of shares as stipulated in the Schedule 1 and 2 to RBI Notification No. FEMA.20/2000-RB dated May 3, 2000. For the details of the conditions specified in the Government Approval, see the section titled “Government Approvals” on page 182. Prohibition by SEBI Our Bank, our Directors, our subsidiaries, our affiliates/associates, and companies with which our Directors are associated with as directors or promoters, have not been prohibited from accessing or operating in the capital markets under any order or direction passed by SEBI. Eligibility for the Issue As a corresponding new bank set up under the Bank Acquisition Act, we are exempt from the eligibility norms specified under clause 2.2 and 2.3 of the SEBI Guidelines to make a public issue of equity shares. The relevant extract of the SEBI Guidelines is set out below: “ 2.4 Exemption from Eligibility Norms 2.4.1 The provisions of clauses 2.2 and 2.3 shall not be applicable in case of; ... (ii) a corresponding new bank set up under the Banking Companies (Acquisition and Transfer of Undertaking) Act, 1970, Banking Companies (Acquisition and Transfer of Undertaking) Act, 1980, State Bank of India Act, 1955 and State Bank of India (Subsidiary Banks) Act, 1959 (hereinafter referred to as “public sector banks”).” Clause 2.2 referred in the clause above relates to unlisted companies and Clause 2.3 relates to listed companies. Clause 2.3 relating to listed companies is reproduced below: 7 “2.3. Public Issue by Listed Companies 2.3.1 A listed company shall be eligible to make a public Issue of equity shares or any other security which may be converted into or exchanged with equity shares at a later date. Provided that the aggregate of the proposed Issue and all previous issues made in the same financial year in terms of size (i.e. offer through offer document + firm allotment + promoters’ contribution through the offer document), Issue size does not exceed 5 times its pre-Issue networth as per the audited balance sheet of the last financial year. Provided that in case there is a change in the name of the issuer company within the last 1 year reckoned from the date of filing of the offer document), the revenue accounted for by the activity suggested by the new name is not less than 50% of its total revenue in the preceding 1 full-year period) 2.3.2. A listed company which does not fulfill the conditions given in the provisos to Clause 2.3.1 above, shall be eligible to make a public Issue subject to complying with the conditions specified in Clause 2.2.2)” Therefore, since our Bank is a corresponding bank and is exempt under clause 2.4 of the SEBI Guidelines, we are eligible to make this Issue. Disclaimer Clause AS REQUIRED, A COPY OF THE RED HERRING PROSPECTUS HAS BEEN SUBMITTED TO SEBI. IT IS TO BE DISTINCTLY UNDERSTOOD THAT SUBMISSION OF THE RED HERRING PROSPECTUS TO SEBI SHOULD NOT, IN ANY WAY, BE DEEMED OR CONSTRUED THAT THE SAME HAS BEEN CLEARED OR APPROVED BY SEBI. SEBI DOES NOT TAKE ANY RESPONSIBILITY EITHER FOR THE FINANCIAL SOUNDNESS OF ANY SCHEME OR THE PROJECT FOR WHICH THE ISSUE IS PROPOSED TO BE MADE OR FOR THE CORRECTNESS OF THE STATEMENTS MADE OR OPINIONS EXPRESSED IN THE RED HERRING PROSPECTUS. THE BOOK RUNNING LEAD MANAGERS, ICICI SECURITIES LIMITED, DSP MERRILL LYNCH LIMITED, ENAM FINANCIAL CONSULTANTS PRIVATE LIMITED, JM MORGAN STANLEY PRIVATE LIMITED AND KOTAK MAHINDRA CAPITAL COMPANY LIMITED, HAVE CERTIFIED THAT THE DISCLOSURES MADE IN THE RED HERRING PROSPECTUS ARE GENERALLY ADEQUATE AND ARE IN CONFORMITY WITH SEBI (DISCLOSURE AND INVESTOR PROTECTION) GUIDELINES, 2000 AS FOR THE TIME BEING IN FORCE. THIS REQUIREMENT IS TO FACILITATE INVESTORS TO TAKE AN INFORMED DECISION FOR MAKING AN INVESTMENT IN THE PROPOSED ISSUE. IT SHOULD ALSO BE CLEARLY UNDERSTOOD THAT WHILE THE BANK IS PRIMARILY RESPONSIBLE FOR THE CORRECTNESS, ADEQUACY AND DISCLOSURE OF ALL RELEVANT INFORMATION IN THE RED HERRING PROSPECTUS, THE BOOK RUNNING LEAD MANAGERS ARE EXPECTED TO EXERCISE DUE DILIGENCE TO ENSURE THAT THE BANK DISCHARGES ITS RESPONSIBILITY ADEQUATELY IN THIS BEHALF AND TOWARDS THIS PURPOSE, THE BOOK RUNNING LEAD MANAGERS, ICICI SECURITIES LIMITED, DSP MERRILL LYNCH LIMITED, ENAM FINANCIAL CONSULTANTS PRIVATE LIMITED, JM MORGAN STANLEY PRIVATE LIMITED AND KOTAK MAHINDRA CAPITAL COMPANY LIMITED AND HAVE FURNISHED TO SEBI, A DUE DILIGENCE CERTIFICATE DATED JANUARY 14, 2005 IN ACCORDANCE WITH THE SEBI (MERCHANT BANKERS) REGULATIONS, 1992 WHICH READS AS FOLLOWS: “WE HAVE EXAMINED VARIOUS DOCUMENTS INCLUDING THOSE RELATING TO LITIGATION LIKE COMMERCIAL DISPUTES, PATENT DISPUTES, DISPUTES WITH COLLABORATORS ETC. AND OTHER MATERIALS IN CONNECTION WITH THE FINALISATION OF THE RED HERRING PROSPECTUS PERTAINING TO THE SAID ISSUE. ON THE BASIS OF SUCH EXAMINATION AND THE DISCUSSIONS WITH THE BANK, ITS DIRECTORS AND OTHER OFFICERS, OTHER AGENCIES, INDEPENDENT VERIFICATION OF THE STATEMENTS CONCERNING THE OBJECTS OF THE ISSUE, PROJECTED PROFITABILITY, PRICE JUSTIFICATION AND THE CONTENTS OF THE DOCUMENTS MENTIONED IN THE ANNEXURE AND OTHER PAPERS FURNISHED BY THE BANK, WE CONFIRM THAT: (A) THE RED HERRING PROSPECTUS FORWARDED TO SEBI IS IN CONFORMITY WITH THE DOCUMENTS, MATERIALS AND PAPERS RELEVANT TO THE ISSUE; (B) ALL THE LEGAL REQUIREMENTS CONNECTED WITH THE SAID ISSUE AS ALSO THE GUIDELINES, INSTRUCTIONS, ETC. ISSUED BY SEBI, THE GOVERNMENT AND ANY OTHER COMPETENT AUTHORITY IN THIS BEHALF HAVE BEEN DULY COMPLIED WITH; (C) THE DISCLOSURES MADE IN THE RED HERRING PROSPECTUS ARE TRUE, FAIR AND ADEQUATE TO ENABLE THE 8 INVESTORS TO MAKE A WELL-INFORMED DECISION AS TO THE INVESTMENT IN THE PROPOSED ISSUE; (D) BESIDES OURSELVES, ALL THE INTERMEDIARIES NAMED IN THE RED HERRING PROSPECTUS ARE REGISTERED WITH SEBI AND THAT TILL DATE SUCH REGISTRATIONS ARE VALID; AND (E) WHEN UNDERWRITTEN, WE SHALL SATISFY OURSELVES ABOUT THE WORTH OF THE UNDERWRITERS TO FULFIL THEIR UNDERWRITING COMMITMENTS.” THE WRITTEN CONSENT FROM PROMOTERS I.E. GOVERNMENT OF INDIA IS BEING OBTAINED FOR INCLUSION OF THEIR SECURITIES AS PART OF PROMOTERS’ CONTRIBUTION SUBJECT TO LOCK-IN AND THAT THE SECURITIES PROPOSED TO FORM PART OF PROMOTERS’ CONTRIBUTION SUBJECT TO LOCK-IN, WILL NOT BE DISPOSED/SOLD/ TRANSFERRED BY THE PROMOTERS. THE SAME IS EXPECTED SOON AND THE RED HERRING PROSPECTUS WILL CARRY THE DETAILS OF THE LOCK-IN. ALL LEGAL REQUIREMENTS PERTAINING TO THE ISSUE WILL BE COMPLIED WITH AT THE TIME OF FILING OF THE RED HERRING PROSPECTUS WITH THE DESIGNATED STOCK EXCHANGE IN ACCORDANCE WITH APPLICABLE LAW, AS ALSO ANY GUIDELINES, INSTRUCTIONS, ETC. ISSUED BY SEBI, GOI AND ANY OTHER COMPETENT AUTHORITY. ALL LEGAL REQUIREMENTS PERTAINING TO THE ISSUE WILL BE COMPLIED WITH AT THE TIME OF REGISTRATION OF THE PROSPECTUS WITH THE DESIGNATED STOCK EXCHANGE IN ACCORDANCE WITH APPLICABLE LAW, AS ALSO ANY GUIDELINES, INSTRUCTIONS, ETC. ISSUED BY SEBI, GOI AND ANY OTHER COMPETENT AUTHORITY.” THE FILING OF THE RED HERRING PROSPECTUS DOES NOT, HOWEVER, ABSOLVE THE BANK FROM ANY LIABILITIES IN THE NATURE OF LIABILITIES UNDER SECTION 63 AND SECTION 68 OF THE COMPANIES ACT OR FROM THE REQUIREMENT OF OBTAINING SUCH STATUTORY AND OTHER CLEARANCES AS MAY BE REQUIRED FOR THE PURPOSE OF THE PROPOSED ISSUE. SEBI FURTHER RESERVES THE RIGHT TO TAKE UP AT ANY POINT OF TIME, WITH THE BOOK RUNNING LEAD MANAGERS, ANY IRREGULARITIES OR LAPSES IN THE RED HERRING PROSPECTUS. Note: Our Bank, our Directors and the BRLMs accept no responsibility for statements made otherwise than in this Red Herring Prospectus or in any advertisements or any other material issued by or at instance of the above mentioned entities and anyone placing reliance on any other source of information, including our website, www.pnbindia.com, would be doing so at his or her own risk. The BRLMs accept no responsibility, save to the limited extent as provided in the Memorandum of Understanding entered into among the BRLMs and us dated January 13, 2005 and the Underwriting Agreement to be entered into among the Underwriters and us. All information shall be made available by us and the BRLMs to the public and investors at large and no selective or additional information would be available for a section of the investors in any manner whatsoever including at road show presentations, in research or sales reports or at bidding centres. Disclaimer in Respect of Jurisdiction This Issue is being made in India to Persons resident in India, including Indian nationals resident in India who are majors, HUFs, companies, corporate bodies and societies registered under the applicable laws in India and authorised to invest in shares, Indian mutual funds registered with SEBI, Indian financial institutions, commercial banks, regional rural banks, co-operative banks (subject to RBI permission), or trusts under the applicable trust law and who are authorised under their constitution to hold and invest in shares, permitted insurance companies and pension funds and to permitted non residents including FIIs, Eligible NRIs and other eligible foreign investors. This Red Herring Prospectus does not, however, constitute an offer to sell or an invitation to subscribe to Equity Shares offered hereby in any other jurisdiction to any Person to whom it is unlawful to make an offer or invitation in such jurisdiction. Any Person into whose possession this Red Herring Prospectus comes is required to inform himself or herself about and to observe, any such restrictions. Any dispute arising out of this Issue will be subject to the jurisdiction of appropriate court(s) in New Delhi, India only. No action has been or will be taken to permit a public offering in any jurisdiction where action would be required for that purpose, except that this Red Herring Prospectus has been filed with SEBI for observations and SEBI has given its observations. 9 Accordingly, the Equity Shares, represented thereby may not be offered or sold, directly or indirectly, and this Red Herring Prospectus may not be distributed, in any jurisdiction, except in accordance with the legal requirements applicable in such jurisdiction. Neither the delivery of this Red Herring Prospectus nor any sale hereunder shall, under any circumstances, create any implication that there has been no change in our affairs from the date hereof or that the information contained herein is correct as of any time subsequent to this date. Accordingly, the Equity Shares are only being offered or sold in the United States to “Qualified Institutional Buyers” as defined in Rule 144A under the U.S. Securities Act of 1933 (the “Securities Act”) and outside the United States to certain Persons in offshore transactions in compliance with Regulation S under the Securities Act. Disclaimer Clause of the BSE As required, a copy of this Red Herring Prospectus has been submitted to BSE. BSE has vide its letter dated January 28, 2005, given permission to the Bank to use BSE’s name in this Red Herring Prospectus as one of the stock exchanges on which our further securities are proposed to be listed. BSE has scrutinised this Red Herring Prospectus for its limited internal purpose of deciding on the matter of granting the aforesaid permission to us. BSE does not in any manner: (i) warrant, certify or endorse the correctness or completeness of any of the contents of this Red Herring Prospectus; or (ii) warrant that this Bank’s securities will be listed or will continue to be listed on BSE; or (iii) take any responsibility for the financial or other soundness of this Bank, its promoters, its management or any scheme or project of this Bank; and it should not for any reason be deemed or construed to mean that this Red Herring Prospectus has been cleared or approved by BSE. Every Person who desires to apply for or otherwise acquires any securities of this Bank may do so pursuant to independent inquiry, investigation and analysis and shall not have any claim against BSE whatsoever by reason of any loss which may be suffered by such Person consequent to or in connection with such subscription/acquisition whether by reason of anything stated or omitted to be stated herein or for any other reason whatsoever. Disclaimer Clause of the NSE As required, a copy of this Red Herring Prospectus has been submitted to NSE. NSE has given vide its letter dated January 18, 2005 permission to us to use NSE’s name in this Red Herring Prospectus as one of the stock exchanges on which our further securities are proposed to be listed, subject to the Bank fulfilling the various criteria for listing of its Equity Shares offerred as part of this Issue. The NSE has scrutinised this Red Herring Prospectus for its limited internal purpose of deciding on the matter of granting the aforesaid permission to us. It is to be distinctly understood that the aforesaid permission given by NSE should not in any way be deemed or construed to mean that this Red Herring Prospectus has been cleared or approved by NSE; nor does it in any manner warrant, certify or endorse the correctness or completeness of any of the contents of this Red Herring Prospectus; nor does it warrant that our securities will be listed or will continue to be listed on the NSE; nor does it take any responsibility for the financial or other soundness of this Bank, its promoters, its management or any scheme or project of this Bank. Every Person who desires to apply for or otherwise acquires any of our securities may do so pursuant to independent inquiry, investigation and analysis and shall not have any claim against NSE whatsoever by reason of any loss which may be suffered by such Person consequent to or in connection with such subscription/acquisition whether by reason of anything stated or omitted to be stated herein or any other reason whatsoever. Filing A copy of this Red Herring Prospectus has been filed with SEBI at Corporation Finance Department, Ground Floor, Mittal Court, “A” Wing, Nariman Point, Mumbai 400 021. A copy of the Red Herring Prospectus, along with the documents required to be filed under applicable law, will be delivered for registration to the Designated Stock Exchange and a copy of the Prospectus, along with the documents required to be filed under applicable law, will be delivered for registration to the Designated Stock Exchange. 10 Listing Our existing Equity Shares are listed on the BSE and NSE. We had applied for voluntary delisting of our Equity Shares from the DSE vide letter dated October 5, 2004 pursuant to a shareholder resolution dated July 3, 2004. We have received a letter dated October 13, 2004 bearing reference number DSE/LIST/R/96 from the DSE informing the Bank that the securities of the Bank are delisted from the DSE with immediate effect. Applications have been made to the BSE and NSE for permission for further listing of our Equity Shares. NSE shall be the Designated Stock Exchange with which the Basis of Allocation shall be finalised for the Non-Institutional Portion and the Retail Portion. If the permission to deal in and for an official quotation of the Equity Shares are not granted by any of the Stock Exchanges, our Bank shall forthwith repay, without interest, all moneys received from the applicants in pursuance of this Red Herring Prospectus. If such money is not repaid within eight days after our Bank becomes liable to repay it (i.e., from the date of refusal or within 70 days from the date of Issue Closing Date, whichever is earlier), then our Bank shall, on and from expiry of eight days, be jointly and severally liable to repay the money, with interest at the rate of 15% per annum on application money. Our Bank shall ensure that all steps for the completion of the necessary formalities for listing and commencement of trading at the Stock Exchanges are taken within seven working days of finalisation and adoption of the Basis of Allotment for the Issue. Impersonation Attention of the applicants is specifically drawn to the following: Any Person who: (a) makes in a fictitious name, an application to a bank for acquiring or subscribing for, any shares therein, or (b) otherwise induces a bank to allot, or register any transfer of shares therein to him, or any other Person in a fictitious name, shall be liable for consequences as prescribed by applicable law. Minimum Subscription If we do not receive the minimum subscription of 90% of the Net Issue to the public to the extent of the amount payable on application, including devolvement of Underwriters, if any, within 60 days from the Bid Closing Date, we shall forthwith refund the entire subscription amount received. If there is a delay beyond eight days after we become liable to pay the amount (i.e., 60 days from the Bid Closing Date), we shall pay interest at the rate of 15% per annum. Withdrawal of the Issue Our Bank, in consultation with the BRLMs, reserves the right not to proceed with the Issue at anytime after the Bid Closing Date, without assigning any reason thereof. Letters of Allotment or Refund Orders We shall give credit to the beneficiary account with Depository Participants within two working days from the date of the finalisation of basis of allocation. We shall ensure despatch of refund orders, if any, of value up to Rs.1,500 by “Under Certificate of Posting”, and shall dispatch refund orders above Rs.1,500, if any, by registered post or speed post at the sole or First Bidder’s sole risk within 15 days of the Bid Closing Date/Issue Closing Date. In accordance the requirements of the Stock Exchanges and SEBI Guidelines, we undertake that: ● Allotment shall be made only in dematerialised form within 15 days from the Issue Closing Date; ● Despatch of refund orders shall be done within 15 days from the Issue Closing Date; and ● We shall pay interest at 15% per annum (for any delay beyond the 15 day time period as mentioned above), if Allotment is not made, refund orders are not despatched and/or demat credits are not made to Bidders within the 15 day time prescribed above, provided that the beneficiary particulars relating to such Bidders as given by the Bidders is valid at the time of the upload of the demat credit. 11 We will provide adequate funds required for despatch of refund orders or allotment advice to the Registrar to the Issue. Refunds will be made by cheques, pay orders or demand drafts drawn on the Escrow Collection Bank and payable at par at places where Bids are received. The bank charges, if any, for encashing such cheques, pay orders or demand drafts at other centres will be payable by the Bidders. Bid/Issue Programme Bidding Period / Issue Period BID / ISSUE OPENS ON : MONDAY, MARCH 7, 2005 BID / ISSUE CLOSES ON : FRIDAY, MARCH 11, 2005 Bids and any revision in Bids shall be accepted only between 10 a.m. and 3 p.m. (Indian Standard Time) during the Bidding Period/Issue Period as mentioned above at the bidding centres mentioned on the Bid cum Application Form except that on the Bid Closing Date, the Bids shall be accepted only between 10 a.m. and 1 p.m. (Indian Standard Time) and uploaded till such time as permitted by the BSE and NSE. The Price Band will be decided by us in consultation with the BRLMs and advertised at least one day prior to the Bid Opening Date/Issue Opening Date in Business Standard, an English language newspaper with wide circulation and Hindustan, a Hindi language newspaper with wide circulation, and includes any revisions thereof. With regard to the Price Band/Floor Price, Bidders are advised to be guided by the price of our listed Equity Shares in the secondary market. For details see the section titled “History and Certain Corporate Matters- Stock Market Data” on page 65. The Bank reserves the right to revise the Price Band during the Bidding Period/Issue Period in accordance with SEBI Guidelines. The cap on the Price Band should not be more than 20% of the floor of the Price Band. Subject to compliance with the immediately preceding sentence, the floor of the Price Band can move up or down to the extent of 20% of the floor of the Price Band disclosed in the Red Herring Prospectus. In case of revision in the Price Band, the Bidding Period/Issue Period will be extended for three additional days after revision of Price Band subject to the Bidding Period/Issue Period not exceeding 13 days. Any revision in the Price Band and the revised Bidding Period/Issue Period, if applicable, will be widely disseminated by notification to BSE and NSE by issuing a press release, and also by indicating the change on the websites of the BRLMs and at the terminals of the Syndicate. BOOK RUNNING LEAD MANAGERS ICICI Securities Limited ICICI Centre, H.T. Parekh Marg, Churchgate, Mumbai, 400 020. Tel : (91 22) 2288 2460 Fax : (91 22) 2282 6580 E-mail : pnb_fpo@isecltd.com DSP Merrill Lynch Limited Mafatlal Centre, 10th Floor, Nariman Point, Mumbai 400 021. Tel: (91 22) 5632 8000 Fax: (91 22) 2204 8518 E-mail: pnb_ fpo@ml.com 12 Enam Financial Consultants Private Limited 801/802, Dalamal Towers, Nariman Point, Mumbai 400 021. Tel : (91 22) 5638 1800 Fax : (91 22) 2284 6824 E-mail: pnb.fpo@enam.com JM Morgan Stanley Private Limited 141, Maker Chambers III, Nariman Point, Mumbai 400 021. Tel : (91 22) 1600 22 0004/5630 3030 Fax : (91 22) 5630 1694 E-mail: pnbfpo@jmmorganstanley.com Kotak Mahindra Capital Company Limited Bakhtawar, 3rd Floor, 229, Nariman Point, Mumbai 400 021. Tel : (91 22) 5634 1100 Fax : (91 22) 2284 0492 E-mail: pnb.fpo@kotak.com CO-MANAGERS A. K. Capital Services Ltd. Flat No. « N », Sagar Apartments, 6, Tilak Marg, New Delhi 110 001 Tel : (91 11) 2338 5704 Fax : (91 11) 2338 5189 E-mail: akcap@bom9.vsnl.net.in BOB Capital Markets Limited Ground Floor, Noble Chambers (Vatsa House) 20-C/D. S. A. Brelvi Road, Fort, Mumbai 400 001 Tel : (91 22) 2284 8492 Fax : (91 22) 2284 5208 E-mail: bobcaps@mtnl.net.in , bobcaps@vsnl.com Centrum Finance Ltd. Khetan Bhavan, 5th Floor, 198, J. Tata Road, Churchgate, Mumbai 400 020. Tel : (91 22) 2202 3838 Fax : (91 22) 2204 6096 E-mail: info@centrum.co.in 13 IDBI Capital Market Services Limited 8th Floor, Bhaktawar, 229 Nariman Point Mumbai 400 021 Tel : (91 22) 5637 1212 Fax : (91 22) 2288 5850 RR Financial Consultants Limited Indraprakash Building 21, Barakhamba Road, New Delhi- 110 001 Tel : (91 11) 2335 2496 Fax : (91 11) 2335 3703 Statement of Inter Se Allocation of Responsibilities for the Issue The following table sets forth the distribution of responsibility and coordination for various activities amongst the BRLMs: Activities Responsibility Co-ordinator Capital structuring with the relative components and formalities ISEC, DSPML, ENAM, ISEC such as type of instruments, etc. JMMS and KOTAK Due diligence of the Bank’s operations/ management/ business plans/ legal, etc. Drafting and design of offer document and of statutory advertisement ISEC, DSPML, ENAM, ISEC including memorandum containing salient features of the Prospectus. JMMS and KOTAK The BRLMs shall ensure compliance with stipulated requirements and completion of prescribed formalities with the Stock Exchanges and SEBI including finalisation of the Prospectus and filing with the Stock Exchanges. Drafting and approval of all publicity material other than statutory ISEC, DSPML, ENAM, ENAM advertisement as mentioned above including corporate JMMS and KOTAK advertisement, brochure, etc. Appointment of other Intermediaries: ISEC, DSPML, ENAM, (a) Printer: ENAM (a) Printers; JMMS and KOTAK (b) Syndicate: ENAM (b) Syndicate Members; (c) Registrar and (c) Registrar and advertising agency; and Advertising (d) Banker to the Issue. Agency: JMMS (d) Banker to the Issue: KOTAK Domestic institutions/banks/mutual funds marketing strategy: ISEC, DSPML, ENAM, KOTAK Finalise the list and division of investors for one on one JMMS and KOTAK meetings, institutional allocation International institutional marketing strategy, roadshow ISEC, DSPML, ENAM, DSPML marketing presentation: JMMS and KOTAK Finalise the list and division of investors for one on one meetings, institutional allocation 14 Activities Responsibility Co-ordinator Retail/Non institutional marketing strategy which will cover, inter alia, ISEC, DSPML, ENAM, ENAM Finalize media, marketing and public relation strategy, JMMS and KOTAK Finalize centers for holding conferences for brokers, etc. Finalize collection centers, Follow-up on distribution of publicity and Issue material including form, Prospectus and deciding on the quantum of the Issue material Managing the Book, coordination with Stock Exchanges, pricing and ISEC, DSPML, ENAM, JMMS allocation to QIB Bidders. JMMS and KOTAK Post bidding activities including management of Escrow Accounts, ISEC, DSPML, ENAM, JMMS co-ordinate non-institutional allocation, intimation of allocation and JMMS and KOTAK dispatch of refunds to Bidders, etc. The post issue activities of the Issue will involve essential follow up ISEC, DSPML, ENAM, JMMS steps, which include finalization of trading and dealing instruments JMMS and KOTAK and dispatch of certificates and demat delivery of shares, with the various agencies connected with the work such as Registrars to the Issue, Banker to the Issue and the bank handling refund business. The BRLMs shall be responsible for ensuring that these agencies fulfill their functions and enable them to discharge this responsibility through suitable agreements with the issuer Bank. SYNDICATE MEMBERS ICICI Brokerage Services Limited ICICI Centre, H.T. Parekh Marg, Churchgate, Mumbai 400 020. Tel: (91 22) 2288 2460 Fax: (91 22) 2282 6580 DSP Merrill Lynch Limited Mafatlal Centre, 10th Floor, Nariman Point, Mumbai 400 021. Tel: (91 22) 5632 8000 Fax: (91 22) 2204 8518 Enam Securities Private Limited 2nd Floor, Khatau Building, 44, Bank Street, off Shaheed Bhagat Singh Road, Fort, Mumbai 400 001. Tel: (91 22) 2267 7901 Fax: (91 22) 2266 5613 JM Morgan Stanley Retail Services Private Limited 141, Maker Chambers III, Nariman Point, Mumbai 400 021. Tel: (91 22) 5630 3030 Fax: (91 22) 5630 1689 15 Kotak Securities Limited Bakhtawar, 1st Floor, 229, Nariman Point, Mumbai 400 021. Tel: (91 22) 5634 1100 Fax: (91 22) 5630 3927 HEAD OFFICE OF OUR BANK Punjab National Bank 7, Bhikhaiji Cama Place, New Delhi 110 066, India. Tel: (91 11) 2610 2303 Fax: (91 11) 2610 8741 COMPANY SECRETARY AND COMPLIANCE OFFICER Ms. Malathi Mohan, Share Department, 5, Sansad Marg, New Delhi 110 001. Tel : (91 11) 2332 3657 Fax : (91 11) 2371 1663 E-Mail: hosd@pnb.co.in Investors can contact the Compliance Officer in case of any pre-Issue or post-Issue related problems such as non-receipt of letters of allotment, credit of allotted shares in the respective beneficiary account, refund orders etc. REGISTRAR TO THE ISSUE MCS Limited, Sri Padmavati Bhavan Plot No. 93, Road No. 16, MIDC Area, Andheri (East) Mumbai 400 092 Tel : (91 22) 2820 1785 Fax : (91 22) 2820 1783 Email: pnbfpo@mcsind.com LEGAL ADVISORS TO THE ISSUE DOMESTIC LEGAL COUNSEL TO THE BANK Amarchand & Mangaldas & Suresh A. Shroff & Co., Amarchand Towers, 216, Okhla Industrial Estate, Phase – III, New Delhi 110 020. Tel : (91 11) 2692 0500 Fax : (91 11) 2692 4900 16 INTERNATIONAL LEGAL COUNSEL TO THE BANK (Advising on matters pertaining to the laws of the State of New York and the Federal law of the United States of America) Cravath, Swaine & Moore LLP, Worldwide Plaza, 825 Eighth Avenue, New York, New York 10019, United States of America. Tel: (1 212) 474 1000 Fax: (1 212) 474 3700 INTERNATIONAL LEGAL COUNSEL TO THE UNDERWRITERS Herbert Smith, Exchange House, Primrose Street, London EC2A 2HS, United Kingdom. Tel: (44) 20 7374 8000 Fax: (44) 20 7374 0888 ADVISORS TO THE ISSUE PNB Gilts Limited, 5, Sansad Marg, New Delhi 110 001 Tel: (91 11) 2332 5759 Fax: (91 11) 2332 5763 AUDITORS M/s Surendar K. Jain & Co, Chartered Accountants, 8/2B, 3rd Floor, WEA, Abdul Aziz Marg, Karol Bagh, New Delhi 110 005. Tel: (91 11) 2574 6190 Fax: (91 11) 2576 6248 M/s. Mookherjee Biswas & Pathak, Chartered Accountants, 5&6 Fancy Lane, 5th Floor, Kolkata 700 001. Tel: (91 33) 2248 1733 Fax: (91 33) 2248 0080 M/s. M. C. Bhandari & Co., Chartered Accountants, 2nd Floor, Room no. 205, 4, Synagogue Street, Kolkata 700 001. Tel: (91 33) 2242 6077 Fax: (91 33) 2274 2444 17 M/s. P. K. Chopra & Co., Chartered Accountants, N- Block, Bombay Life Building, Above Post Office, Connaught Circus, New Delhi 110 001. Tel: (91 11) 2331 2869 Fax: (9111) 2331 4442 M/s. Ramanlal G. Shah & Co., Chartered Accountants, Shreeji House, 4th Floor, Behind M.J. Library, Ellis Bridge, Ahmedabad 380 006. Tel: (91 79) 2657 8819 Fax: (91 79) 2657 5401 M/s. B.K. Ramadhyani & Co., Chartered Accountants, 4B Chitrapur Bhavan, No 68, 8th Main, 15th Crossroad, Malleswaram, Bangalore 560 055. Tel: (91 80) 2334 4295 Fax: (91 80) 2334 8964 Banker to the Issue and Escrow Collection Bank Punjab National Bank, Branch Office: 5, Sansad Marg, New Delhi 110 001. Credit Rating As the Issue is of equity shares, credit rating is not required. Trustees As the Issue is of equity shares, the appointment of trustees is not required. Book Building Process Book building refers to the process of collection of Bids, on the basis of the Red Herring Prospectus within the Price Band. The Issue Price is fixed after the Bid Closing Date/Issue Closing Date. The principal parties involved in the Book Building Process are: (1) The Bank; (2) Book Running Lead Managers; (3) Syndicate Members who are intermediaries registered with SEBI or registered as brokers with BSE/NSE and eligible to act as Underwriters. Syndicate Members are appointed by the BRLMs; (4) Escrow Collection Bank; and (5) Registrar to the Issue. SEBI through its guidelines has permitted an issue of securities to the public through 100% Book Building Process, wherein: (i) upto 50% of the Net Issue to the public shall be allocated on a discretionary basis to QIBs (ii) not less than 25% of the Net Issue 18 to the public shall be available for allocation on a proportionate basis to the Non-Institutional Bidders (iii) not less than 25% of the Net Issue to the public shall be available for allocation on a proportionate basis to Retail Individual Bidders, subject to valid Bids being received at or above the Issue Price. However, as intimated by us to SEBI vide letter dated February 2, 2005, and noted by them in their letter dated February 4, 2005, the Net Issue to the public shall be allocated in the following proportion: upto 50% shall be allocated to QIBs, at least 15% shall be allocated to Non-Institutional Bidders and atleast 35% shall be allocated to Retail Individual Bidders. Pursuant to amendments to the SEBI Guidelines, QIB Bidders are not allowed to withdraw their Bid (s) after the Bid Closing Date/Issue Closing Date and for further details please refer to the section titled “Terms of the Issue” on page 217. The Bank shall comply with guidelines issued by SEBI for this Issue. In this regard, our Bank has appointed ICICI Securities Limited, DSP Merrill Lynch Limited, Enam Financial Consultants Private Limited, JM Morgan Stanley Private Limited and Kotak Mahindra Capital Company Limited as the BRLMs to manage the Issue and to procure subscription to the Issue. The process of book building, under SEBI Guidelines, is relatively new and the investors are advised to make their own judgment about investment through this process of book building prior to making a Bid in the Issue. Illustration of Book Building and Price Discovery Process (Investors should note that this example is solely for illustrative purposes and is not specific to the Issue) Bidders can bid at any price within the price band. For instance, assume a price band of Rs. 20 to Rs. 24 per share, issue size of 3,000 equity shares and receipt of five bids from bidders out of which one bidder has bid for 500 shares at Rs. 24 per share while another has bid for 1,500 shares at Rs. 22 per share. A graphical representation of the consolidated demand and price would be made available at the bidding centres during the bidding period. The illustrative book as shown below shows the demand for the shares of the company at various prices and is collated from bids from various investors. Bid Quantity Bid Price (Rs.) Cumulative Quantity Subscription 500 24 500 27.77% 1000 23 1500 83.33% 1500 22 3000 166.67% 2000 21 5000 277.78% 2500 20 7500 416.67% The price discovery is a function of demand at various prices. The highest price at which the issuer is able to issue the desired number of shares is the price at which the book cuts off i.e. Rs. 22 in the above example. The issuer, in consultation with the book running lead managers, will finalise the issue price at or below such cut off price, i.e., at or below Rs. 22. All bids at or above this issue price and cut-off bids are valid bids and are considered for allocation in the respective categories. Steps to be taken for bidding: 1. Check eligibility for bidding, please refer to the section titled “Issue Procedure - Who Can Bid?” on page 223; 2. Ensure that the Bidder has a demat account; and 3. Ensure that the Bid cum Application Form is duly completed as per instructions given in this Red Herring Prospectus and in the Bid cum Application Form. 4. Ensure that the Bid cum Application Form is accompanied by the PAN, or by Form 60 or Form 61 as may be applicable together with necessary documents providing proof of address. See page 236 for details. Bidders are specifically requested not to submit their GIR number instead of the PAN number as the Bid is liable to be rejected. Underwriting Agreement After the determination of the Issue Price and allocation of our Equity Shares but prior to filing of the Prospectus with the Designated Stock Exchange, our Bank will enter into an Underwriting Agreement with the Underwriters for the Equity Shares proposed to be offered through this Issue. It is proposed that pursuant to the terms of the Underwriting Agreement, the BRLMs 19 shall be responsible for bringing in the amount devolved in the event that their respective Syndicate Members do not fulfill their underwriting obligations. The Underwriters have indicated their intention to underwrite the following number of Equity Shares: Name and Address of the Underwriters Indicative Number of Amount Equity Shares to be Underwritten Underwritten (Rs. million) ICICI Securities Limited 15,999,920 [●] ICICI Centre, H.T.Parekh Marg, Churchgate, Mumbai, 400020 DSP Merrill Lynch Limited 15,999,920 [●] Mafatlal Centre, 10th Floor, Nariman Point, Mumbai 400 021 Enam Financial Consultants Private Limited 8,500,000 [●] 801/802, Dalamal Towers Nariman Point Mumbai 400 021 JM Morgan Stanley Private Limited 15,999,920 [●] 141, Maker Chambers III, Nariman Point, Mumbai 400 021. Kotak Mahindra Capital Company Limited 15,999,920 [●] Bakhtawar, 3rd Floor, 229 Nariman Point, Mumbai 400 021. ICICI Brokerage Services Limited 100 [●] ICICI Centre, H.T.Parekh Marg, Churchgate, Mumbai, 400020. Enam Securities Private Limited 7,500,020 [●] 2nd Floor, Khatau Building, 44, Bank Street, Off Shaheed Bhagat Singh Road, Fort, Mumbai- 400 001. JM Morgan Stanley Retail Services Private Limited 100 [●] 141, Maker Chambers III, Nariman Point, Mumbai 400 021 Kotak Securities Limited, 100 [●] Bakhtawar, 1st floor, 229, Nariman Point Mumbai- 400 021. The above-mentioned amount is indicative underwriting and this would be finalized after pricing and actual allocation. The Underwriting Agreement is dated [●]. 20 In the opinion of the Board of Directors acting through the Chairman and Managing Director or the Executive Director, based on certificates dated [●] given to them by the BRLMs and the Syndicate Members, the resources of the Underwriters are sufficient to enable them to discharge their respective underwriting obligations in full. All the above-mentioned Underwriters are registered with SEBI under Section 12(1) of the SEBI Act or registered as brokers with the Stock Exchanges. The above Underwriting Agreement has been accepted by the Board of Directors acting through the Chairman and Managing Director or the Executive Director of our Bank and our Bank has issued letters of acceptance to the Underwriters. Allocation among Underwriters may not necessarily be in proportion to their underwriting commitments. Notwithstanding the above table, the Underwriters shall be severally responsible for ensuring payment with respect to Equity Shares allocated to investors procured by them. In the event of any default, the respective Underwriter in addition to other obligations to be defined in the Underwriting Agreement, will also be required to procure/subscribe to the extent of the defaulted amount. Allocation to QIB Bidders is discretionary as per the terms of this Red Herring Prospectus and may not be proportionate in any way and the patterns of allocation to the QIB Bidders could be different for the various Underwriters. 21 CAPITAL STRUCTURE Share capital as of the date of filing of the Red Herring Prospectus with SEBI (before and after the Issue and without and with giving effect to the Return of Proceeds and the Capital Reduction) is set forth below: (Rs.) Aggregate Aggregate Value nominal value at Issue Price A. Authorised Capital 1 1,500,000,000Equity Shares of Rs. 10 each 15,000,000,000 B. Issued, Subscribed and Paid-Up Capital before the Issue 265,302,500Equity Shares of Rs.10 each fully paid-up 2,653,025,000 C. Present Issue in terms of this Red Herring Prospectus Issue of 80,000,000Equity Shares of Rs. 10 each fully paid-up 800,000,000 Out of which (I) 8,000,000 Equity Shares reserved for Employees on a competitive basis 80,000,000 (II) 8,000,000 Equity Shares reserved for Existing Shareholders on a competitive basis 80,000,000 (III) Net Issue to the public 64,000,000 Equity Shares of Rs. 10 each 640,000,000 [●] D. Equity Capital after the Issue but before the Capital Reduction 345,302,500Equity Shares of Rs.10 each fully paid-up 3,453,025,000 [●] F. Capital Reduction Capital Reduction of 30,000,000 Equity Shares of Rs. 10 each fully paid-up, of the President of India in our Bank. (300,000,000) G. Equity Capital after the Issue and the Capital Reduction 315,302,500Equity Shares of Rs.10 each fully paid-up 3,153,025,000 H. Share Premium Account Before the Issue 1,114,285,200 After the Issue without giving effect to the Return of Proceeds and the Capital Reduction [●] After the Issue, giving effect to the Return of Proceeds and the Capital Reduction [●] 1 The authorized share capital of our Bank has remained 1,500,000,000 Equity Shares of Rs. 10 each since the nationalisation of the Bank on July 19, 1969. ● The President of India acting through the MoF, holds 80% of the issued and paid up equity capital of our Bank. After the Issue and before the Capital Reduction, the shareholding of the Promoter will be at least 61.47% of the fully diluted post Issue paid up equity share capital of our Bank. However, following the Issue and the Capital Reduction the shareholding of 22 the Promoter will be at least 57.8% of the fully diluted post-Issue paid up equity share capital of the Bank. The Promoter vide letter no. F.No. 11/29/2004-BOA dated January 20, 2005 issued by the Government of India, MoF, Department of Economic Affairs (Banking Division) has given its approval for lock in of 20% of the fully diluted post Issue paid up equity share capital of our Bank for a period of three years from the date of Allotment. Our Board of Directors had authorised a fresh issue of up to 50,000,000 Equity Shares pursuant to a resolution passed at its meeting held on September 11, 2004. Our shareholders subsequently authorised the fresh issue of up to 50,000,000 Equity Shares, by a resolution passed unanimously at the general meeting of our Bank held on October 11, 2004, subject to the approval of the Government, the RBI, SEBI and other applicable authorities. The shareholder resolution gave the Board the authority and power to accept any modification in the proposal as may be required or imposed by such authorities and as agreed to by the Board. Our Bank applied to the Government on October 14, 2004 for its sanction to a fresh issue of up to 50,000,000 Equity Shares. By its letter bearing number F.No.11/29/2004-BOA dated January 7, 2005, pursuant to Section 3 (2B) (c) of the Bank Acquisition Act, the Department of Economic Affairs, MoF, GoI conveyed its approval on inter alia the terms that the Issue size to be increased to 80,000,000 Equity Shares and as a condition to the approval, the amount raised for 30,000,000 Equity Shares through the public offer should be returned to the Government at the Issue Price by March 31, 2005. Further, in terms of the Government Approval, the proportionate cost of public issue to the extent of 30,000,000 Equity Shares, is to be borne by the Government. Further, in accordance with the provisions of Section 3(2BBA)(a) of the Bank Acquisition Act, the shareholders of the Bank, at an annual general meeting held on August 4, 2003, resolved to return capital to the GoI to the extent of 130,000,000 Equity Shares or such other number of Equity Shares in one or more tranches, and authorized the Board to accept or agree to any modifications in the amount, number of shares or manner of reduction of paid up share capital. This resolution has not been modified or cancelled and permits our Board of Directors to accept the condition of the Government requiring the Capital Reduction. Following the receipt of the Government Approval pursuant to the authority granted to the Board by the shareholders of our Bank in the extraordinary general meeting dated September 11, 2004, our Board, by its resolution dated January 13, 2005, resolved to accept the condition of the Government Approval that the amount raised through the issue of 30,000,000 Equity Shares out of the total issue of 80,000,000 Equity Shares would be returned to the Government by March 31, 2005, and also resolved that the Bank would reduce 30,000,000 Equity Shares held by the President of India. Accordingly, we shall transfer the amount raised through the issue of 30,000,000 Equity Shares in this Issue to the Government by March 31, 2005. For further details relating to the Capital Reduction, see sections titled “Risk Factors” on page x, “General Information - Authority for the Issue” on page 7, “Objects of the Issue” on page 29, “Government Approvals” on page 182 and “Material Contracts and Documents for Inspection” on page 278. We shall debit an amount equal to the nominal value of 30,000,000 Equity Shares from our share capital account. Against return of the nominal value of 30,000,000 Equity Shares we will cancel 30,000,000 Equity Shares held by the President of India in our Bank. In addition, we shall debit the difference between the value of 30,000,000 Equity Shares at the Issue Price raised through the Issue and the nominal value of 30,000,000 Equity Shares from our share premium account. In terms of the Government Approval, the proportionate cost of public issue to the extent of 30,000,000 Equity Shares is to be borne by the Government. Although the Bank Acquisition Act permits us to reduce the share capital of the Government, it is ambiguous on whether it permits reduction from the share premium account. Although we believe that the Bank Acquisition Act permits such reduction to be made from the share premium account and we have obtained independent opinions to such effect, in the event the Bank Acquisition Act were interpreted differently by statutory authorities, it is possible that the part of the payment to the Government which represents the amount debited from the share premium account could be deemed to be a dividend and consequently, there could be tax implications for us. We have received opinions from independent tax experts that there are no accounting or legal issues relating to the payment of the difference between the amount raised at the Issue Price for 30,000,000 million Equity Shares and the nominal value of 30,000,000 million Equity Shares from the share premium account and as the Government is not a taxable entity capable of being considered as an assessee, the amount raised for 30,000,000 Equity Shares through the Issue, which has to be returned to GoI at the Issue Price by March 31, 2005, is not subject to dividend tax. 23 However, as abundant caution to avoid any impact of tax liability on our Bank in this regard, vide letter dated February 7, 2005, we have communicated to the Government that in the event any such tax liability arises on the Bank, the Government should either facilitate a waiver of such tax implications or as a proportionate cost of the Issue to be borne by Government, indemnify the Bank for the same. As on the date hereof, the proposal is still pending before the Government. For further details on the experts’ opinions on tax and the letter dated February 7, 2005 written by our Bank to the Government, see the section titled “Material Contracts and Documents for Inspection” on page 278. ● Pursuant to the sanction received from the Government of India, MoF, Department of Economic Affairs (Banking Division) vide their letter no. F.No. 12/7/97-BOA dated November 13, 1997, the equity share capital of the Bank was restructured by returning Rs.1383.3 million on November 15, 1997 being part of the capital received from the GoI on January 1, 1994, against redemption of an equivalent amount of 10% recapitalisation bonds subscribed to by the Bank on November 15, 1997. ● We have received permission from the Foreign Exchange Department, RBI vide their letter no.FE.CO.FID/4998/ 10.02.40(8435)/2004-05 dated February 2, 2005 permitting us to issue shares to NRIs / FIIs with repatriation benefits out of our public Issue of 80,000,000 Equity Shares. The RBI has imposed the condition that post Issue the non resident equity holding in our Bank should not exceed 20% of the paid up capital. The permission is further subject to the conditions laid down by the Government of India in their approval letter no. F.No.11/29/2004-BOA dated January 7, 2005, condition prescribed/stipulated by SEBI in this connection and the terms and conditions for issue of shares as stipulated in the Schedule 1 and 2 to RBI Notification No. FEMA.20/2000-RB dated May 3, 2000. Notes to the Capital Structure 1. Share Capital History of our Bank since nationalisation on July 19, 1969: Date/Year of No. of Face Issue Consideration Reasons for Cumulative Cumulative Allotment Shares Value Price Allotment Share Paid-up (in (Rs.) (Rs.) Premium Share Capital million) (Rs. in million) July 19, 1969 2 10 10 Nil 20 December 31, 1981 3 10 10 other than cash capitalisation of reserves Nil 50 December 31, 1982 2.5 10 10 Cash allotment of shares to the Nil 75 President of India acting through the MoF against release of funds by GoI December 31, 1983 2.5 10 10 other than cash capitalisation of reserves Nil 100 December 31, 1985 38 10 10 other than cash allotment of shares to the Nil 480 President of India acting through the MoF in lieu of 7.75% special securities of GoI December 31, 1986 52 10 10 other than cash allotment of shares to the Nil 1000 President of India acting through the MoF in lieu of special securities of GoI December 31, 1990; 12.84 10 10 Cash allotment of shares to the Nil 1128.40 December 31, 1991; President of India acting December 31, 1992 through MoF in lieu of financial assistance for industrial export project from World Bank 24 Date/Year of No. of Face Issue Consideration Reasons for Cumulative Cumulative Allotment Shares Value Price Allotment Share Paid-up (in (Rs.) (Rs.) Premium Share Capital million) (Rs. in million) March 31, 1993 75 10 10 other than cash allotment of shares to Nil 1878.40 the President of India acting through the MoF in lieu of 7.75% special securities of GoI March 31, 1994 415 10 10 other than cash allotment of shares to the Nil 6028.40 President of India acting through the MoF in lieu of 10% recapitalisation bonds 2006 of GoI March 31, 1994 0.21 10 10 other than cash allotment of shares to the Nil 6030.5 President of India acting through MoF in lieu of financial assistance for industrial export project from the World Bank March 31, 1994 (239.43) 10 10 other than cash adjustment for the capital Nil 3636.20 of New Bank of India March 31, 1997 (13.05) 10 10 other than cash transfer of capital to the Nil 3505.70 reserves and surplus earlier increased by allotment of shares in lieu of World bank assistance November 15, 1997 (138.33) 10 10 other than cash return of capital to GoI Nil 2122.40 and redemption of equal amount of 10% recapitalisation bonds April 20, 2002 53.06 10 31 Cash allotment of shares to the 1114.28 2653.02 applicants under initial issue of capital 2. Promoter’s contribution and lock-in (a) Details of Promoter’s contribution locked in for three years are as follows: Name of Date of Date when Consideration No. of % of % of Lock-in Shareholder Allotment/ made fully Equity Pre- Post- Period Acquisition paid-up Shares Issue Issue in the (Face paid- paid- previous Value up up issue Rs10/-) Capital Capital President of India March 31, March 31, Other than 69,060,500* 26.03% 20% 3 years acting through the 1994 1994 cash* MoF These shares were allotted in lieu of 10% recapitalisation bonds received from GoI. 25 (b) Details of Promoter’s contribution locked in for one year are as follows: The Equity Shares of our Bank are listed on the BSE and NSE. Our Promoter is not participating in this Issue, hence, there is no requirement for lock in of the Equity Shares other than those locked in for three years in (a) above. (c) The shareholding of the Promoter and the public before and after the Issue is as follows: Before the Issue After the Issue without After the Issue with Capital Reduction Capital Reduction Equity Shares % Equity Shares % Equity Shares % Promoters 212,241,300 80.00 212,241,300 61.47 182,241,300 57.80 Public 53,061,200 20.00 133,061,200 38.53 133,061,200 42.20 Total 265,302,500 100 345,302,500 100 315,302,500 100 (d) The list of top ten shareholders of our Bank and the number of Equity Shares held by them is as under: i. Top ten shareholders of our Bank as on February 10, 2005 the date of filing of this Red Herring Prospectus with the Designated Stock Exchange are as follows: Sl.No. Name of Shareholders Number of shares 1. President of India 212,241,300 2. Goldman Sachs Investments (Mauritius) I Limited 5,801,627 3. Morgan Stanley and Co. International Limited A/C Morgan Stanley Dean 5,577,277 Witter Mauritius 4. UBS Securities Asia Limited A/c Swiss Finance Corporation (Mauritius) Limited 4,028,166 5. CLSA Merchant Bankers Limited A/c Calyon 3,732,018 6. Copthall Mauritius Investment Limited 3,445,372 7. Sanford C Bernstien and Co., Delaware Business Trust- Emerging Markets Value Series 2,459,000 8. Emerging Markets Management, L. L. C. A/c EMSAF- Mauritius 2,428,663 9. Alliance Capital Management LP A/c Sanford C Bernstein Fund, INC 2,375,000 10. The State Teacher Retirement System of Ohio 1,150,000 Total 243, 238, 423 26 ii. Top ten shareholders of our Bank as on February 4, 2005, ten days before the date of filing of this Red Herring Prospectus with the Designated Stock Exchange are as follows: Sl.No. Name of Shareholders Number of shares 1 President of India 212,241,300 2 Morgan Stanley and Co. International Limited A/C Morgan Stanley Dean Witter Mauritius 6,230,077 3 Goldman Sachs Investments (Mauritius) I Limited 5,802,890 4 UBS Securities Asia Limited A/c Swiss Finance Corporation (Mauritius) Limited 4,031,586 5 CLSA Merchant Bankers Limited A/c Calyon 3,732,018 6 Copthall Mauritius Investment Limited 3,445,322 7 Emerging Markets Management, L.L.C A/C EMSAF- Mauritius 2,428,663 8. Alliance Capital Management LP A/c Sanford C Bernstein Fund, INC 2,375,000 9 Sanford C Bernstien and Co., Delaware Business Trust – Emerging Markets Value Series 1,844,000 10 The State Teacher Retirement System of Ohio 1,150,000 Total 243,280,856 iii. Top ten shareholders as on February 14, 2003, i.e., two years prior to date of filing of this Red Herring Prospectus with Designated Stock Exchange is as follows: Sl.No. Name of Shareholders Number of shares 1 President of India 212,241,300 2 Alliance Capital Mutual Fund A/C Alliance Equity Fund 1,968,043 3 India Liberalisation Fund (Mauritius) Limited 1,600,000 4 Zurich Trustee Company (I) Private Limited A/C Zurich India Mutual Fund- Zurich India Equity 1,450,000 5 Prudential ICICI Trust –Growth Plan 1,385,510 6. Birla Sunlife Trustee Company Limited A/c Birla Mutual Fund-Birla Advantage 1,000,600 7. Emerging Markets Management, LLC A/c EMSAF-Mauritius 989,925 8. Templeton Mutual Fund A/C Franklin India Blue Chip Fund 926,272 9. Birla Sunlife Trustee Company Limited A/c Birla Mutual Fund-Birla Balance 790,566 10 Zurich Trustee Company (I) Private Limited A/C Zurich India Mutual Fund- Zurich India 600,000 Total 222,952,216 iv. There are no outstanding warrants, options or rights to convert debentures, loans or other instruments into our Equity Shares. v. Neither the Promoter, nor our Directors have purchased or sold any Equity Shares, directly or indirectly, during a period of six months preceding the date on which this Red Herring Prospectus is filed with SEBI. Our Bank, our 27 Directors and the BRLMs have not entered into any buy-back and/or standby arrangements for purchase of Equity Shares from any Person. vi. In the Net Issue to the public, in case of over-subscription in all categories, up to 50% of the Net Issue to the public shall be available for allocation on a discretionary basis to Qualified Institutional Buyers, a minimum of 15% of the Net Issue to the public shall be available for allocation on a proportionate basis to Non-Institutional Bidders and a minimum of 35% of the Net Issue to the public shall be available for allocation on a proportionate basis to Retail Individual Bidders, subject to valid Bids being received at or above the Issue Price. Under-subscription, if any, in any portion would be met with spill over from other categories at the sole discretion of our Bank in consultation with the BRLMs. vii. A total of up to 10% of the Issue size, i.e., up to 8,000,000 Equity Shares, has been reserved for allocation to the Employees on a proportionate basis, subject to valid Bids being received at or above the Issue Price and subject to the maximum Bid in this portion being 500 Equity Shares. Only Employees on the rolls of the Bank as on January 1, 2005 would be eligible to apply in this Issue under reservation for our Employees. Employees may bid in the Net Issue to the public portion as well and such Bids shall not be treated as multiple Bids. Any under subscription in the Equity Shares under the Employees Reservation Portion would be treated as part of the Net Issue to the public in the manner detailed in the section titled “Issue Procedure” on page 223. viii. A total of up to 10% of the Issue size, i.e., up to 8,000,000 Equity Shares, has been reserved for allocation to the Existing Shareholders on a proportionate basis, subject to valid Bids being received at or above the Issue Price. Only Existing Shareholders as on February 11, 2005 would be eligible to apply in this Issue under reservation for our Existing Shareholders. Existing Shareholders may bid in the Net Issue to the public portion as well and such Bids shall not be treated as multiple Bids. Any under subscription in the Equity Shares under the Existing Shareholders Reservation Portion would be treated as part of the Net Issue to the public in the manner detailed in the section titled “Issue Procedure” on page 223. ix. An investor in the Net Issue to the public cannot make a Bid for more than the number of Equity Shares offered in the Net Issue. This is further subject to the maximum limit of investment prescribed under relevant laws applicable to each category of investor. Further an investor applying in the category reserved for the Existing Shareholders or Employees cannot make a Bid for more than the number of Equity Shares offered in the respective category. x. There would be no further issue of capital whether by way of issue of bonus shares, preferential allotment, rights issue or in any other manner during the period commencing from submission of this Red Herring Prospectus with SEBI until the Equity Shares have been listed. xi. Other than the Capital Reduction, we presently do not intend or propose to alter our capital structure for a period of six months from the date of opening of the Issue, by way of split or consolidation of the denomination of Equity Shares or further issue of Equity Shares (including issue of securities convertible into or exchangeable, directly or indirectly for Equity Shares) whether preferential or otherwise, except that we may issue options to our employees pursuant to any employee stock option scheme or, if we enter into acquisitions or joint ventures, we may, subject to necessary approvals, consider raising additional capital to fund such activity or use Equity Shares as currency for acquisition or participation in such joint ventures. xii. There shall be only one denomination of the Equity Shares, unless otherwise permitted by law. We shall comply with such disclosure and accounting norms as may be specified by SEBI from time to time. xiii. As on February 4, 2005 the total number of holders of Equity Shares is 59,928. xiv. The Bank has not raised any bridge loans against the proceeds of the Issue. xv. We have not issued any Equity Shares out of revaluation reserves. We have also not issued any shares or debentures for consideration other than cash other than mentioned elsewhere in this Red Herring Prospectus, within the two years preceding the date of this Red Herring Prospectus. xvi. Other than the Capital Reduction, we have not entered into standby, buyback or similar arrangements for this Issue. xvii.We have not made any preferential allotments or bonus issues after listing pursuant to our IPO but prior to filing of the Red Herring Prospectus. 28 OBJECTS OF THE ISSUE Objects of the Issue The RBI, which regulates us, requires us to maintain a minimum capital to risk weighted assets ratio of 9.0%, at least half of which should consist of Tier 1 capital. Our total capital adequacy ratio based on our restated financial statements was 11.91% and our Tier 1 capital adequacy ratio was 7.00% as of March 31, 2003, 12.79% and 6.70% as of March 31, 2004 and 12.45% and 7.31% as of September 30, 2004. The requirements for Tier 1 capital and total capital adequacy ratios are expected to increase with the proposed implementation of the Basel II standards in late 2006 or early 2007 and we will need to augment our capital base to meet these ratios. If the Basel II standards were applied on a pro forma basis as of September 30, 2004, we would expect our total capital adequacy ratio to be 9.85%, compared to 12.58% on a historical, unrestated basis. Further, we would require additional capital for future growth. The objects of the Issue are to augment our capital base to meet the future capital requirements arising out of the implementation of the Basel II standards and the growth in our assets, primarily our loan and investment portfolio due to the growth of the Indian economy and for other general corporate purposes including meeting the expenses of the Issue. As we are engaged in the business of banking and our subsidiaries are engaged in banking and other financial services, we are seeking to strengthen our capital base to support the future growth in our assets and comply with the capital adequacy requirements applicable to us. Other general corporate purposes would include development of infrastructure to support our business growth and service our customers. An additional object of the Issue is to comply with the conditions specified in the Government Approval letter, which has been accepted by our Board by their resolution dated January 13, 2005, as a result of which our Board has resolved that the amount raised through the issue of 30,000,000 Equity Shares out of the total issue of 80,000,000 Equity Shares would be returned to the Government by March 31, 2005, and that the Bank would reduce 30,000,000 shares held by the President of India in accordance with the provisions of Section 3(2BBA)(a) of the Bank Acquisition Act. Accordingly, we shall transfer the amount raised through the issue of 30,000,000 Equity Shares in this Issue to the Government by March 31, 2005. We shall debit an amount equal to the nominal value of 30,000,000 Equity Shares from our share capital account. Against return of the nominal value of 30,000,000 Equity Shares we will cancel 30,000,000 Equity Shares held by the President of India in our Bank. In addition, we shall debit the difference between the value of 30,000,000 Equity Shares at the Issue Price raised through the Issue and the nominal value of 30,000,000 Equity Shares from our share premium account. In terms of the Government Approval, the proportionate cost of public issue to the extent of 30,000,000 Equity Shares is to be borne by the Government. For further details relating to the Capital Reduction, see the sections titled “Risk Factors” on page x, “General Information - Authority for the Issue” on page 7, “Capital Structure” on page 22, “Government Approvals” on page 182 and “Material Contracts and Documents for Inspection” on page 278. Utilization of Issue Proceeds The proceeds of the Issue will be utilized for the purposes described under “Objects of the Issue” above. Our Constitutional Documents enable us to undertake our existing activities and the activities for which the funds are being raised by us in the Issue. Requirement and Sources of Funds Requirement of Funds (In Rs. million) Augment our capital base to meet our future capital adequacy requirements arising out the proposed implementation of the Basel II standards, the general growth of our business and for other general corporate purposes. [●] Return of capital to the Government of India as explained above [●] Estimated Issue expenses [●] Total [●] 29 Sources of Funds (In Rs. million) Proceeds of the Issue [●] Total [●] Will be updated in the Prospectus. Estimated Issue Expenses Activity (In Rs. million) Estimated lead management, underwriting and selling commission 0.60% of the Issue size(1) [●] Estimated other expenses (including advertising fee, registrars fee, legal fee, printing and stationery, distribution, cost of road shows and listing fee) 138.80 Total [●] (1) Subject to approval of the RBI. 30 THE INDIAN BANKING SECTOR The information in this section has been extracted from publicly available documents from various sources, including officially prepared materials from the GoI and its various ministries and the RBI, and has not been prepared or independently verified by us or any of our advisors. Wherever we have relied on figures published by the RBI, unless stated otherwise, we have relied on the RBI Annual Report, 2003-2004 and Quarterly Statistics on Deposits and Credit of Scheduled Commercial Banks as of September 2004. THE INDIAN BANKING SYSTEM History The evolution of the modern commercial banking industry in India can be traced to 1786 with the establishment of Bank of Bengal in Calcutta. Three presidency banks were set up in Calcutta, Bombay and Madras. In 1860, the limited liability concept was introduced in banking, resulting in the establishment of joint stock banks like Allahabad Bank Limited, Punjab National Bank Limited, Bank of Baroda Limited and Bank of India Limited. In 1921, the three presidency banks were amalgamated to form the Imperial Bank of India , which took on the role of a commercial bank, a bankers’ bank and a banker to the government. The establishment of the RBI as the central bank of the country in 1935 ended the quasi-central banking role of the Imperial Bank of India. In order to serve the economy in general and the rural sector in particular, the All India Rural Credit Survey Committee recommended the creation of a state-partnered and state sponsored bank taking over the Imperial Bank of India and integrating with it, the former state-owned and state-associate banks. Accordingly, the State Bank of India (“SBI”) was constituted in 1955. Subsequently in 1959, the State Bank of India (Subsidiary Bank) Act was passed, enabling the SBI to take over eight former state-associate banks as its subsidiaries. In 1969, 14 private banks were nationalised followed by six private banks in 1980. Since 1991 many financial reforms have been introduced substantially transforming the banking industry in India. Reserve Bank of India The RBI is the central banking and monetary authority in India. The RBI manages the country’s money supply and foreign exchange and also serves as a bank for the GoI and for the country’s commercial banks. In addition to these traditional central banking roles, the RBI undertakes certain developmental and promotional activities. The RBI issues guidelines, notifications, circulars on various areas including exposure standards, income recognition, asset classification, provisioning for non-performing assets, investment valuation and capital adequacy standards for commercial banks, long-term lending institutions and non-banking finance companies. The RBI requires these institutions to furnish information relating to their businesses to the RBI on a regular basis. Commercial Banks Commercial banks in India have traditionally focused on meeting the short-term financial needs of industry, trade and agriculture. At the end of September 2004, there were 291 scheduled commercial banks in the country, with a network of 67,221 branches. Scheduled commercial banks are banks that are listed in the second schedule to the Reserve Bank of India Act, 1934, and may further be classified as public sector banks, private sector banks and foreign banks. Industrial Development Bank of India was converted into a banking company by the name of Industrial Development Bank of India Ltd. with effect from October, 2004 and is a scheduled commercial bank. Scheduled commercial banks have a presence throughout India, with nearly 70.1% of bank branches located in rural or semi-urban areas of the country. A large number of these branches belong to the public sector banks. Public Sector Banks Public sector banks make up the largest category of banks in the Indian banking system. There are 27 public sector banks in India. They include the SBI and its associate banks and 19 nationalised banks. Nationalised banks are governed by the Banking Companies (Acquisition and Transfer of Undertakings) Act 1970 and 1980. The banks nationalised under the Banking Companies (Acquisition and Transfer of Undertakings) Act 1970 are referred to as ‘corresponding new banks’. Punjab National Bank is a public sector bank nationalised in 1969 and a corresponding new bank under the Bank Acquisition Act. At the end of September 2004, public sector banks had 46,782 branches and accounted for 74.9% of the aggregate deposits and 70.5% of the outstanding gross bank credit of the scheduled commercial banks. 31 Regional Rural Banks Regional rural banks were established from 1976 to 1987 jointly by the Central Government, State Governments and sponsoring public sector commercial banks with a view to develop the rural economy. Regional rural banks provide credit to small farmers, artisans, small entrepreneurs and agricultural labourers. There were 196 regional rural banks at the end of September 2004 with 14,4430 branches, accounting for 3.6% of aggregate deposits and 3.0% of gross bank credit outstanding of scheduled commercial banks. Private Sector Banks After the first phase of bank nationalisation was completed in 1969, the majority of Indian banks were public sector banks. Some of the existing private sector banks, which showed signs of an eventual default, were merged with state-owned banks. In July 1993, as part of the banking reform process and as a measure to induce competition in the banking sector, the RBI permitted entry by the private sector into the banking system. This resulted in the introduction of nine private sector banks. These banks are collectively known as the ‘‘new’’ private sector banks. There are nine “new” private sector banks operating at present. Foreign Banks At the end of June 2004, there were 32 foreign banks with 217 branches operating in India, accounting for 4.6% of aggregate deposits and 7.0% of outstanding gross bank credit of scheduled commercial banks. The Government of India permits foreign banks to operate through (i) branches; (ii) a wholly owned subsidiary or (iii) a subsidiary with aggregate foreign investment of up to 74% in a private bank. The primary activity of most foreign banks in India has been in the corporate segment. However, some of the larger foreign banks have made consumer financing a significant part of their portfolios. These banks offer products such as automobile finance, home loans, credit cards and household consumer finance. The GoI in 2003 announced that wholly- owned subsidiaries of foreign banks would be permitted to incorporate wholly-owned subsidiaries in India. Subsidiaries of foreign banks will have to adhere to all banking regulations, including priority sector lending norms, applicable to domestic banks. In March 2004, the Ministry of Commerce and Industry, GoI announced that the foreign direct investment limit in private sector banks has been raised to 74% from the existing 49% under the automatic route including investment by FIIs. The announcement also stated that the aggregate of foreign investment in a private bank from all sources would be allowed up to a maximum of 74% of the paid up capital of the bank. The RBI notification increasing the limit to 74% is however still awaited. Cooperative Banks Cooperative banks cater to the financing needs of agriculture, small industry and self-employed businessmen in urban and semi-urban areas of India. The state land development banks and the primary land development banks provide long-term credit for agriculture. In light of the liquidity and insolvency problems experienced by some cooperative banks in fiscal 2001, the RBI undertook several interim measures to address the issues, pending formal legislative changes, including measures related to lending against shares, borrowings in the call market and term deposits placed with other urban cooperative banks. The RBI is currently responsible for supervision and regulation of urban co-operative societies, the National Bank for Agriculture and Rural Development, state co-operative banks and district central co-operative banks. The Banking Regulation (Amendment) and Miscellaneous Provisions Bill, 2003, which was introduced in the Parliament in 2003, proposed the regulation of all co-operative banks by the RBI. The Bill has not yet been ratified by the Indian Parliament and is not in force. Term Lending Institutions Term lending institutions were established to provide medium-term and long-term financial assistance to various industries for setting up new projects and for the expansion and modernization of existing facilities. These institutions provide fund-based and non-fund based assistance to industry in the form of loans, underwriting, direct subscription to shares, debentures and guarantees. The primary long-term lending institutions include Industrial Development Bank of India (converted into a banking company with effect from October, 2004), IFCI Ltd., Infrastructure Development Finance Company Limited, Industrial Investment Bank of India and Industrial Credit Corporation of India Limited (prior to its amalgamation). The term lending institutions were expected to play a critical role in industrial growth in India and, accordingly, had access to concessional government funding. However, in recent years, the operating environment of the term lending institutions has changed substantially. Although the initial role of these institutions was largely limited to providing a channel for government 32 funding to industry, the reform process required them to expand the scope of their business activities. Their new activities include: ■ Fee-based activities like investment banking and advisory services; and ■ Short-term lending activity including corporate loans and working capital loans. Pursuant to the recommendations of the Committee on Banking Sector Reforms (Narasimhan Committee II), S.H. Khan Working Group, a working group created in 1999 to harmonise the role and operations of term lending institutions and banks, the RBI, in its mid-term review of monetary and credit policy for fiscal 2000, announced that long-term lending institutions would have the option of transforming themselves into banks subject to compliance with the prudential norms as applicable to banks in India. In April 2001, the RBI issued guidelines on several operational and regulatory issues which were required to be addressed in evolving the path for transition of a term lending institution into a universal bank. Industrial Development Bank of India was converted into a banking company with the name of Industrial Development Bank of India Ltd. within the meaning of the Bank Regulation Act and the Companies Act with effect from October, 2004. It is currently able to carry on banking operations in addition to the business being transacted by it as a term lending institution. LIBERALISATION AND THE REFORM PROCESS Impact of Liberalisation on the Banking Sector Until 1991, the financial sector in India was heavily controlled, and commercial banks and term lending institutions, the two dominant financial intermediaries, had mutually exclusive roles and objectives and operated in a largely stable environment, with little or no competition. Term lending institutions were focused on the achievement of the Indian government’s various socio-economic objectives, including balanced industrial growth and employment creation, especially in areas requiring development. They lending institutions provided access to long-term funds at subsidised rates through loans and equity from the Government of India and from funds guaranteed by the Government of India originating from commercial banks in India and foreign currency resources originating from multilateral and bilateral agencies. The focus of the commercial banks was primarily to mobilise household savings through demand and time deposits and to use these deposits to meet the short-term financial needs of borrowers in industry, trade and agriculture. In addition, the commercial banks provided a range of banking services to individuals and businesses. However, since 1991, there have been comprehensive changes in the Indian financial system. Various financial sector reforms, implemented since 1991, have transformed the operating environment of the banks and long-term lending institutions. In particular, the deregulation of interest rates, the emergence of a liberalised domestic capital market, and entry of new private sector banks, along with the broadening of term lending institutions’ product portfolios, have progressively intensified the competition among banks and term lending institutions. The RBI has permitted the transformation of term lending institutions into banks subject to compliance with the applicable law. Banking Sector Reforms In the wake of the last decade of financial reforms, the banking industry in India has undergone a significant transformation, which has covered almost all important facets of the industry. Most large banks in India were nationalised in 1969 and thereafter were subject to a high degree of control until reform began in 1991. In addition to controlling interest rates and entry into the banking sector, the regulations also channelled lending into priority sectors. Banks were required to fund the public sector through the mandatory acquisition of low interest-bearing government securities or statutory liquidity ratio bonds to fulfil statutory liquidity requirements. As a result, bank profitability was low, non-performing assets were comparatively high, capital adequacy was diminished, and operational flexibility was hindered. Committee on the Financial System ( Narasimham Committee I) The Committee on the Financial System (Narasimham Committee I) was set up in August 1991 to recommend measures for reforming the financial sector. Many of the recommendations made by the committee, which addressed organisational issues, accounting practices and operating procedures, were implemented by the Government of India. The major recommendations 33 that were implemented included the following: ● With fiscal stabilisation and the Government increasingly resorting to market borrowing to raise resources, the statutory liquidity ratio or the proportion of the banks’ net demand and time liabilities that were required to be invested in government securities was reduced from 38.5% in the pre-reform period to 25.0% in October 1997. This meant that the significance of the statutory liquidity ratio shifted from being a major instrument for financing the public sector in the pre-reform era to becoming a prudential requirement; ● Similarly, the cash reserve ratio or the proportion of the bank’s net demand and time liabilities that were required to be deposited with the RBI was reduced from 15.0% in the pre-reform period to 5.0% currently; ● Special tribunals were created to resolve bad debt problems; ● Most of the restrictions on interest rates for deposits were removed and commercial banks were allowed to set their own level of interest rates for all deposits except savings bank deposits; and ● Substantial capital infusion to several state-owned banks was approved in order to bring their capital adequacy closer to internationally accepted standards. The stronger public sector banks were given permission to issue equity to increase capital. Committee on Banking Sector Reform (Narasimham Committee II) The second Committee on Banking Sector Reform (Narasimham Committee II) submitted its report in April 1998. The major recommendations of the committee were in respect of capital adequacy requirements, asset classification and provisioning, risk management and merger policies. The RBI accepted and began implementing many of these recommendations in October 1998. The success of the reforms were aided to a large extent by the relative macroeconomic stability during the period. Another distinguishing feature of the reforms was the successful sequencing and gradual introduction of the reforms. Banks have implemented new prudential accounting norms for classification of assets, income recognition and loan loss provisioning. Following the Bank for International Settlements (BIS) guidelines, capital adequacy norms have also been prescribed. To meet additional capital requirements, public sector banks have been allowed to access the market for funds. Interest rates have been deregulated, while the rigour of directed lending has been progressively reduced. A number of measures have been taken to reduce the level of NPAs like establishment of DRTs, Lok Adalats and the system of One-Time Settlement (OTS) of dues through mutual negotiation. A system of Corporate Debt Restructuring (CDR), based on the “London Approach” has been put in place as a voluntary process of corporate restructuring. For information on CDR see the section titled “The Indian Banking Sector- Corporate Debt Restructuring Forum” on page 35. Recent Structural Reforms Amendments to the Banking Regulation Act Legislation seeking to amend the Banking Regulation Act was introduced in the Parliament in 2003 but has not yet been ratified by the Indian Parliament and is therefore not in force. The main amendments proposed to: ●· Permit banking companies to issue non-redeemable and redeemable preference shares; ● Make prior approval of the RBI mandatory for the acquisition of more than 5.0% of a banking company’s paid up capital by any individual or firm or group; ● Prohibit lending to relatives of directors and to non-subsidiary companies that are under the same management as the banking company, joint ventures, associates or the holding company of the banking company. Lending to directors and to companies with directors common to the banking company is already prohibited; ●· Remove the minimum statutory liquidity ratio requirement of 25.0%, giving the RBI discretion to reduce the statutory liquidity ratio to less than 25.0%; ● Bring mergers of non-bank finance companies with banking companies into the governance of the Indian Banking Regulation Act. Mergers of non-bank finance companies with banking companies are currently governed by the Companies Act. The Banking Regulations (Amendment) and Miscellaneous Provisions Bill, 2003 will, if passed, require mergers of non-bank 34 finance companies with banking companies to be approved by the majority of the shareholders of both companies and by the RBI. It also provides, if the merger is approved, for dissenting shareholders at their option to be paid in exchange for their shares the value of their shares as determined by the RBI; and ● Bring all co-operative banks under the supervision of the RBI. Legislative Framework for Recovery of Debts Due to Banks In fiscal 2003, the Parliament passed the Securitisation Act. The Securitisation Act provides the powers of “seize and desist” to banks. The Act provides that a “secured creditor” may, in respect of loans classified as non-performing in accordance with the RBI guidelines, give notice in writing to the borrower requiring it to discharge its liabilities within 60 days, failing which the secured creditor may take possession of the assets constituting the security for the loan, and exercise management rights in relation thereto, including the right to sell or otherwise dispose of the assets. This Act also provides for the setting up of asset reconstruction companies regulated by the RBI to acquire assets from banks and financial institutions. The constitutionality of the Securitisation Act was challenged in Mardia Chemicals Limited v. Union of India, AIR 2004 SC 2371, a petition filed before the Supreme Court. The Supreme Court upheld the validity of the Act except Section 17(2) wherein they found that the requirement of making a deposit of 75% of the amount claimed at the time of making a petition or an appeal to the DRT under Section 17 in order to challenge the measures taken by the creditor in pursuance of Section 13(4) was unreasonable and therefore, struck down. The RBI has issued guidelines for asset reconstruction companies in respect of their establishment, registration and licensing by the RBI, and operations. Earlier, following the recommendations of the Narasimham Committee I, the Recovery of Debts due to Banks and Financial Institutions Act, 1993 was enacted. This legislation provides for the establishment of a tribunal for speedy resolution of litigation and recovery of debts owed to banks or financial institutions. The legislation creates tribunals before which the banks or the financial institutions can file a suit for recovery of the amounts due to them. However, if a scheme of reconstruction is pending before the Board for Industrial and Financial Reconstruction, under the Sick Industrial Companies (Special Provision) Act, 1985, no proceeding for recovery can be initiated or continued before the tribunals. While presenting its budget for fiscal 2002, the Government of India announced measures for the setting up of more debt recovery tribunals and the eventual repeal of the Sick Industrial Companies (Special Provision) Act, 1985. While the Parliament has repealed this Act, the notification to make the repeal effective has not yet been issued. Corporate Debt Restructuring Forum (CDR) To put in place an institutional mechanism for the restructuring of corporate debt, the RBI has devised a corporate debt restructuring system. The objective of this framework is to ensure a timely and transparent mechanism for the restructuring of corporate debts of viable entities facing problems, outside the purview of the Board for Industrial and Financial Reconstruction, debt recovery tribunals and other legal proceedings. In particular, the framework aims to preserve viable corporates that are affected by certain internal and external factors and minimize the losses to the creditors and other stakeholders through an orderly and coordinated restructuring program. The corporate debt restructuring system is a non-statutory mechanism and a voluntary system based on debtor-creditor and inter-creditor agreements. Any lender having a minimum 20% exposure in term loan or working capital may make a reference to the Corporate Debt Restructuring Forum. The system put in place by the RBI contemplates a three tier structure with the CDR Standing Forum at the helm, which is the general body of all member institutions, out of which is carved out the core group, a niche body of select institutions that decides policy matters. The decisions on restructuring are taken by the CDR Empowered Group, which has all the member banks/FIIs as its members. To assist the CDR Forum in secretarial matters and for analysis of the restructuring packages, a CDR Cell has been formed. The total membership of the CDR Forum, as on March 31, 2004 is 60, of which there are 14 FIIs, 27 public sector banks and 19 private sector banks. Technology Technology is emerging as a key-driver of business in the banking and financial services industry. Banks are developing alternative channels of delivery like ATMs, telebanking, remote access and internet banking etc. Indian banks have been making significant investments in technology. Besides computerization of front-office operations, the banks have moved 35 towards back-office centralization. Banks are also implementing “Core Banking”, which provides connectivity between branches and helps offer a large number of value-added products, benefiting a larger number of customers. The RBI Annual Report for the year 2003-04 states that the use of automated teller machines (“ATMs”) has been growing rapidly and this has helped in optimising the investments made by banks in infrastructure. Banks have joined together in small clusters to share their ATM networks during the year. There are five such ATM network clusters functioning in India. The total number of ATMs installed by the public sector banks stood at 8,219 at end March 2004 as compared with 5,963 ATMs at the end of March 2003. The payment and settlement system is also being modernised. The Reserve Bank of India is actively pursuing the objective of establishing a Real Time Gross Settlement (RTGS) system, on par with other developed economies. Corporate Governance Adoption of good corporate governance practices has been getting the attention of banks as well as the regulators and owners in India. Banks in India now typically have an audit committee of the board of directors which is entrusted with the task of overseeing the organisation, operationalisation and quality control of the internal audit function, reviewing financial accounts and follow-up with the statutory and external auditors of the bank as well as examinations by regulators. Disclosure levels in bank balance sheets have been enhanced, while measures have also been initiated to strengthen corporate governance in banks. Consolidation Indian banks are increasingly recognizing the importance of “size”. The consolidation efforts have received encouragement from the views publicly expressed recently by the Government favouring consolidation in the Indian banking sector. Although there have been instances of mergers, these have usually involved financially distressed banks. Mergers and acquisitions are seen banks as a means of achieving inorganic growth in size and attaining economies of scale and scope. Notwithstanding the government ownership of public sector banks, the government has indicated that it would not stand in the way of mergers of public sector banks, provided the bank boards come up with a proposal of merger, based on synergies and potential for improved operational efficiency. Further, the government has also provided tax breaks aimed at promoting mergers and acquisitions (Section 72 (A) of the I.T. Act enables the acquiring entity (which could be a company, a corresponding new bank, a banking company or a specified bank) the benefit of “carry forward and set-off of accumulated losses and unabsorbed depreciation” of the acquired entity, subject to specified conditions being fulfilled). It is envisaged that the consolidation process in the public sector bank group is imminent, particularly as banks will be required to attain higher capital standards under Basel II and meet the pressures of competition by adoption of the extended universal banking model. Moving ahead Bank deposits continue to remain an important instrument of financial saving. The share of bank deposits in household savings has shown an increase from 30.8% in fiscal 2000 to 40.5% in fiscal 2004. The increased use of technology should help banks to reduce transaction costs, and enhance cross-selling of bank products. It has been recognized that the agricultural sector has not been a major beneficiary of the decade long reform process and a skewed interest rate structure has emerged in case of agricultural loans vis-à-vis consumer loans, mainly as a result of fierce competitive pressures in the consumer finance segment. The GoI intends to address this underlying weakness, considering that 70% of the population is in India’s villages and the agro-economy needs more infrastructure investment. Accordingly, the GoI’s policy is to double the level of agricultural credit in the next three years and the public sector banks have geared themselves to pursue this objective. This however, is not expected to result in risk concentration as agricultural advances of public sector banks constituted only 15.4% of their net credit at the end of March 2004. Further, being of smaller quantum, agricultural advances help banks to achieve risk-dispersal and it is generally seen that recovery rates have consistently higher. Moreover, banks have been provided tax breaks for boosting agricultural advances (banks can claim income tax exemption on 10% of their average rural advances) and 7.5% of net profit before provisions and tax. More recently, the RBI has also modified the NPA norms for agricultural advances (linking delinquency to the crop cycle). 36 NEW INITIATIVES IN THE BANKING SECTOR Risk Management & Basel II With gradual deregulation, banks are now exposed to different types of risks. In view of the dynamic nature of the financial market, banks face various market risks like interest rate risk, liquidity risk, and exchange risk. In respect of lending, they face credit risk which includes default risk and portfolio risk. Banks also face risks like operational risk. In preparation for the adoption of the Basel II accord, banks have already been required by the Reserve Bank of India to take active measures in terms of risk management systems, evaluate capital charges including for operational risk and bring about more transparency in financial reporting as part of market discipline. The RBI has also moved towards adoption of Risk Based Supervision (RBS) of banks under which the risk profile of the banks will decide their supervisory cycles - a bank with higher risk rating will undergo more frequent supervisory reviews than those with lower risk rating. The RBI has also indicated that it will adopt a phased approach to the implementation of the Basel II. RTGS Implementation in India With the commencement of operations of the Real Time Gross Settlement i.e. RTGS system from March 26, 2004, India crossed a major milestone in the development of systemically important payment systems and complied with the core principles framed by the Bank for International Settlements. It was a ‘soft’ launch with four banks, besides the RBI, as participants. As on March 31, 2004, there are 71 direct participants in the RTGS system, including PNB. The salient features of the RTGS are as follows: ● Payments are settled transaction by transaction for high value and retail payments; ● Settlement of funds is final and irrevocable; ● Settlement is done on a real time basis and the funds settled can be further used immediately; ● It is a fully secure system which uses digital signatures and Public Key Infrastructure (PKI) based inscription for safe and secure message transmission; ● There is a provision for intra-day collaterised liquidity support for member banks to smoothen the temporary mismatch of fund flows; and ● RTGS provides for transfer of funds relating to inter bank settlements as also for customer related fund transfers. More than 75% of the value of inter bank transfers, which was earlier being settled through the deferred net settlement systems (“DNSS”) based inter-bank clearing, is now being settled under RTGS. ANNUAL POLICY STATEMENT The RBI has renamed its credit policy as the “Annual Policy” statement since it is more aimed at structural adjustments rather than controlling the credit flow in the economy. As per a circular issued by the RBI in September, 2004, the rate of CRR of scheduled commercial banks has been increased in two stages, i.e., to 4.75% from September 18, 2004 and 5% from October 2, 2004 of net demand and time liabilities as on the last Friday of the second preceding fortnight. The SLR, bank rate and Repo Rate have not been changed in the Annual Policy of the RBI for the year 2003-04. However, in its mid-term review of the annual policy which was published on October 26, 2004, the RBI increased the repo rate to 4.75% from 4.50%. As part of its effort to continue bank reform, the RBI has announced a series of measures in its monetary and credit policy statements aimed at deregulating and strengthening the financial system. In the mid term review of Annual Policy for the year 2004-05, RBI has stated that the overall stance of monetary policy for the second half of the year shall be as follows: ● Provision of appropriate liquidity to meet credit growth and support investment and export demand in the economy while placing equal emphasis on price stability; ● Consistent with the above, to pursue and interest rate environment that is conductive to macroeconomic and price stability, and maintaining the momentum of growth; and 37 ● To consider measures in a calibrated manner, in response to evolving circumstances with a view to stabilising inflationary expectations. In the monetary and credit policy for fiscal 2005 as amended by the mid term review of Annual Policy for the year 2004-05, the RBI has introduced the following measures, among others: ● There is no change in the bank rate, which remains at 6.0%. The seven-day repo rate has been increased by 25 basis points from 4.50% to 4.75% effective from October 27, 2004. ● Liquidity adjustment facility (“LAF”) scheme: The international usage of “repo” and “reverse repo” terms would be adopted from October 27, 2004. The LAF scheme would be operated with overnight fixed rate repo and reverse repo with effect from November 1, 2004. Accordingly, auctions of 7-day and 14-day repo (‘reverse repo’ in international parlance) would stand discontinued from November 1, 2004. ● Changes in the interest rate policy relating to ceiling on interest rates on Non-Resident (External) Rupee Deposits, permission to banks to fix interest rates on FCNR(B) deposits on monthly basis for the following month based on the rates prevailing as on the last working day of previous month, reduction of tenor of domestic term deposits. ● Measures for the removal of bottlenecks in credit delivery mechanism. These measures include service area approach, priority sector lending (enhanced lending to agriculture and distribution of inputs, enhanced lending to small and marginal farmers, special agriculture credit plans, enhancement of composite loan limit to SSI Units, enhancement of ceiling of housing loans etc.), rural infrastructure development fund, corporate debt restructuring mechanism, liberalisation of bank finance to NBFCs, gold card scheme for exporters. ● Structural and developmental measures for deepening and widening the government securities market. ● Measures for simplifying the systems and procedures for offering better customer service and to continue with the liberalisation process for improvement of the foreign exchange market . 38 BUSINESS Overview We are a leading public sector commercial bank in India, offering banking products and services to corporate and commercial, retail and agricultural customers. We started our operations in 1895 and since then have grown to become India’s third largest bank in terms of assets and second largest bank in terms of number of branches. Although we began our operations in the agriculturally rich areas of Northern India, we have expanded our operations to provide products and services to over 35 million customers across India through over 4,000 branches. Our banking operations for corporate and commercial customers include a range of products and services for large corporate customers as well as for small and middle market businesses and government entities. We cater to the financing needs of the agricultural sector and have created innovative financing products for farmers. We also provide significant financing to other priority sectors including small scale industries. We offer a wide range of retail credit products including home loans, personal loans and automobile loans. Through our subsidiaries and joint ventures, we deal in Indian government securities and provide housing finance and asset management services. Through our treasury operations, we manage our balance sheet, including the maintenance of required regulatory reserves, and seek to maximize profits from our trading portfolio by taking advantage of market opportunities. Since 1969, when we became a public sector bank, we have managed to continue to grow our business while maintaining a strong balance sheet. As of September 30, 2004, our total deposits represented 85.9% of our total liabilities. On average, interest free demand deposits and low interest savings deposits represented 43.8% of these deposits in the first six months of fiscal 2005. These low-cost deposits led to an average cost of funds excluding equity for the first six months of fiscal 2005 of 4.7%. As of September 30, 2004, our gross and net non-performing assets constituted 7.65% and 0.30% of our gross and net advances, respectively. In fiscal 2004 our total income was Rs. 96.5 billion and our net profit was Rs. 11.1 billion before adjustment and Rs. 10.6 billion after adjustment as part of the restatement of our financial statements for this Issue. In the first six months of fiscal 2005 our total income was Rs. 51.9 billion and our net profit was Rs. 7.4 billion before adjustment and Rs. 7.9 billion after adjustment. Between fiscal 2002 and 2004, our total income grew at a compound annual rate of 12.5%, our unadjusted and adjusted net profit grew at a compound annual rate of 40.4% and 37.4%, respectively, and our total deposits and total advances grew at a compound annual growth rate of 17.1% and 17.2%, respectively. We intend to maintain our position as a cost efficient and customer friendly institution that provides comprehensive financial and related services. We seek to achieve this by continuing to adopt technology which will integrate our extensive branch network. We intend to grow by cross-selling various financial products and services to our customers and by expanding geographically in India and internationally. We are committed to excellence in serving the public and also maintaining high standards of corporate responsibility. In line with our philosophy of aiding India’s development we have opened branches in many rural areas. Strategy Our goal is to further strengthen our position as one of India’s premier commercial banks and to increase our profitability by providing a comprehensive range of products and services and superior customer service. Our business strategy emphasizes the following elements: Expand our business as the Indian economy grows by using our extensive domestic branch network to deliver high quality service that is tailored to the needs of our customers The Indian economy is currently experiencing a high rate of growth. The consequent increase in the size and incomes of the middle and upper-income classes has resulted in significant expansion in India’s banking and financial services industry. We believe that we can benefit from the growth of the Indian economy and increase our market share by continuing to emphasize high quality service through our extensive network of over 4,000 branches and our comprehensive product offerings which are tailored to our customers. To this end, we intend to continue to expand and at the same time restructure and rationalize our branch network to make our services more efficient. We also have introduced specialized branches that cater to the specific needs of various categories of customers. We intend to continue to implement these measures so as to ensure that we have one of the largest, as well as one of the most efficient, delivery networks in India. 39 Strengthen and expand our corporate and commercial franchise We seek to build on our historical strength in the corporate and commercial sector by expanding our product and service offerings to large corporate customers and government entities as well as growth-oriented small and medium sized businesses. We seek to increase our customer base by offering innovative products and servicing the needs of our corporate and commercial customers through specialized branches, including those targeted at large and medium sized corporate customers and those targeted at small scale industries. Additionally, we seek to cross-sell our fee-based products and services to our current customer base and thereby increase our income from these products and services. Expand retail opportunities by effectively leveraging our customer base and increasing our product offerings With the increase in household income levels in India and the consequent need for diversified financial services, the retail sector has emerged as a rapidly growing opportunity for banks with the skills and infrastructure to adequately service this market. The keys to our retail strategy are wide distribution, convenient customer service, a full range of products and prudent risk management. Cross-selling of a wide range of banking services and insurance, credit and investment products to our customers is a critical aspect of our retail strategy as we leverage our current customer base to expand into new product areas. Maintain and enhance our franchise in the agriculture sector We intend to maintain and enhance our position as one of the leading banks for agricultural lending in India. In 2004, we exceeded the national goal for allocation of credit to the agricultural sector. We have introduced innovative products such as our Krishi credit cards, which provide farmers with convenient financing for their production, investment and consumption activities. We have a nationwide franchise in the agriculture sector, with concentration in northern India, one of the country’s most fertile areas. We intend to further expand our agriculture lending activities in other areas in India, including those which have historically not been adequately served. We also intend to expand our agriculture lending activities into areas such as horticulture and herbal cultivation. Another aspect of our strategy is to further strengthen our ties with the agricultural community by providing training and social support programs for the rural populace. Develop our technological capabilities to interconnect our branch network and improve our services We believe that technology is a critical differentiator in the banking industry and over the past few years have devoted substantial resources to upgrading our technology. We have electronically interconnected over 690 of our branches (as of September 30, 2004), which we believe is significantly more than any other bank in India. We intend to extend this to between 1,500 and 2,000 branches by March 31, 2006. We also intend to expand the delivery channels of our services and have introduced internet banking and set up over 400 ATMs. We aim to improve service by further using interconnectivity to implement centralized credit management, helpdesk services and data backup capabilities. Maintain high asset quality standards through comprehensive risk management We have maintained high quality loan and investment portfolios by carefully targeting our customer base and implementing a comprehensive risk assessment process and diligent risk monitoring and remediation procedures. We apply our credit risk rating process to advances in excess of Rs. 2 million. Additionally, we actively monitor our loans and reassess their credit ratings once a year or more frequently if they are at risk. We also apply aggressive remediation policies to recover non-performing loans. Our provisioning policies for non-performing assets are in accordance with the RBI guidelines. As of September 30, 2004 we had floating provisions of Rs. 13.9 billion for non-performing assets, which was significantly in excess of those required under the RBI guidelines. Our gross NPA coverage ratio was 96.4% as of September 30, 2004. Maintain our low cost of funds We have one of the lowest overall cost of funds in the Indian banking industry. We have achieved this through a large base of low cost deposits, with total deposits representing 90.7% of our funding as of September 30, 2004. Interest free demand deposits and low interest savings deposits constituted 10.3% and 34.9%, respectively, of our total deposits as of September 30, 2004. We believe we can maintain and enlarge this low-cost funding base by leveraging our extensive branch network and our customer base of over 35 million accounts and by offering a wide range of products and high quality customer service. 40 Overview of Our Operations We offer products and services to corporate and commercial customers, as well as agricultural and retail customers. The following table presents our outstanding loans by sector and the proportion of these loans to our outstanding total loans, as of the dates indicated: (Rs. in billions, except for percentages) March 31, 2003 March 31, 2004 September 30, 2004 Sector Loans % of total Loans % of total Loans % of total Corporate and Commercial 265.9 66.1% 287.1 60.8% 311.3 60.0% of which Small Scale Industries (SSIs)(1) 47.9 11.9% 57.0 12.1% 57.3 11.0% Retail 65.6 16.3% 91.5 19.4% 99.7 19.2% Agriculture(1) 70.8 17.6% 93.6 19.8% 107.7 20.8% Total outstanding loans 402.3 100.0% 472.2 100.0% 518.7 100.0% (1) As of the last reporting Friday of the month indicated. We also undertake treasury operations and offer treasury related products and services to our customers. These include interest rate and currency swaps and foreign exchange transactions. Services for Corporate and Commercial Customers General We provide commercial banking products and services to corporate and commercial customers. Our loan products include term loans for the acquisition, construction or improvement of assets as well as short term loans, cash credit, export credit and other working capital financing and bill discounting. We categorize corporate and commercial borrowers by loan size, with small loans being loans under Rs. 35 million, medium loans being loans between Rs. 35 million and Rs. 120 million and large loans being loans over Rs. 120 million. We also provide credit substitutes such as letters of credit and guarantees. In addition, we provide other services such as cash management services and certain capital markets services. We provide commercial banking products and services to corporate and commercial customers, including mid-sized and small businesses and government entities. Most of our top twenty customers in terms of loan exposure are PSUs and government companies. The average size of rated standard accounts in the corporate and commercial sector with Rs. 5 million or more outstanding was Rs. 79.5 million as of September 30, 2004. Corporate and Commercial Sector Lending Activities Term Loans Our term loans consist primarily of financing for the acquisition, construction or improvement of assets, including project finance. Although most of our loans are denominated in rupees, we provide foreign currency loans which amounted to 4.3% of our total portfolio as of September 30, 2004. These loans are typically secured by the real and personal property financed as well as by other assets of the borrower. Repayment is typically in installments over the life of the loan. We also provide short term loans with a maturity of three to twelve months for temporary cash flow and other purposes which are repayable with a bullet payment on maturity. These can be denominated in rupees or other currencies and issued at fixed or floating rates. These loans are usually provided to highly rated corporate customers and may be unsecured. Cash Credit and Other Working Capital Facilities Cash credit facilities are the most common form of working capital financing in India. These are revolving credit facilities secured by working capital assets, such as inventory and receivables. We sometimes take additional security in the form of liens on fixed assets, pledges of marketable securities and personal guarantees. Facilities are typically provided for 12 month periods and bear interest, usually at floating rates. We also provide overdrafts, working capital demand loans, working capital term loans and short term loan facilities to our corporate and commercial borrowers. 41 Bill Discounting Bill discounting involves the financing of short term trade receivables by the issuance of negotiable instruments by the borrower. We discount these instruments and in certain cases, repayment of the bill is assumed by way of a commitment (in the form of a letter of credit ) by another bank. Export Credits The RBI requires banks to make loans to exporters at predetermined rates of interest. We provide credit in rupees as well as foreign currencies for the pre-shipment and post-shipment requirements of exporters. The RBI provides export credit refinancing for an eligible portion of total outstanding export loans at the bank rate prevailing from time to time. We also earn fees and commissions from other fee-based products and services that we offer exporter customers. Other Credit and Financing Products In addition to our lending activities we provide a variety of products to meet the financing needs of our corporate and commercial customers. These include funded products like bridge loans and unfunded products like letters of credit and guarantees. Letters of Credit We provide letter of credit facilities for fees based on the term of the facility and the amount drawn down. The facilities are typically partially or fully secured by assets including cash deposits, documents of credit, stocks and receivables. These facilities are typically given for twelve month periods, often as part of a package of working capital financing products or term loans. Guarantees We issue guarantees on behalf of our customers to guarantee their payment and performance obligations. These are generally secured by account indemnities, a counter guarantee or a fixed or floating charge on the assets of the borrower, including cash deposits. Other Services for Corporate and Commercial Customers We also offer the following fee based services to our corporate and commercial customers: Cash Management Services We offer our corporate and commercial clients collection, payment and remittance services, allowing them to reduce the time period between collections and remittances, thereby streamlining their cash flows. Our cash management products include physical cheque-based clearing in locations where settlement systems are not uniform, electronic clearing services and central pooling of country-wide collections. Our customers pay us fees for these services based on the volume of the transaction, the location of the cheque collection center and the speed of delivery. Capital Markets Products We act as a banker for capital market transactions. These include offerings of equity and debt. The companies involved in these transactions are required to maintain the subscription funds with the bankers to the offering until the allotment and the refund of excess subscription is completed. This process generally takes about 15 to 30 days, resulting in short term deposits with us. We also act as bankers to corporate customers for their interest and dividend payouts, which results in interest-free float balances for us. We also act as the debenture trustee to a number of our corporate customers. Small Scale Industries We provide financing to “small scale industries” or “SSIs”. SSIs are defined as manufacturing, processing and servicing businesses with up to Rs. 50 million invested in plant and machinery for certain industries such as hosiery, hand tools, drugs and pharmaceuticals and stationery items and up to Rs. 10 million invested in plant and machinery for other small scale industries. SSIs are also considered a priority sector for directed lending purposes. See the section titled “Business-Directed Lending” below. As of the last reporting Friday in September 2004, SSI loans constituted 11.3% of our net bank credit. As of the last reporting Friday in September, 2004 we had an outstanding loan portfolio of Rs. 57.3 billion in this segment compared to Rs. 42 48.5 billion as of the last reporting Friday in September 2003, representing growth of approximately 18.1%. We have also received awards and recognition from the Government of India relating to our efforts in financing SSI businesses. Our products for the SSI sector are intended to facilitate the establishment, expansion and modernization of businesses, including acquiring fixed assets, plant and machinery and meeting working capital needs. We target this sector with specifically designed products such as our Laghu Udayami Credit Card and Artisan Credit Card which provide working capital lines up to Rs. 1 million and Rs. 0.2 million, respectively. We provide flexible security requirements to make credit more accessible to SSI borrowers. For manufacturing, service and information technology SSIs, loans of up to Rs. 2.5 million are guaranteed by the Credit Guarantee Fund Trust for Small Industries, which allows us to be more flexible in our security requirements. In order to complement our strengths in the agricultural sector, we have sought out agriculture based SSIs in areas such as food processing which also enable us to provide financing at multiple levels of the supply chain. We aim to offer efficient loan processing and are working with our peer banks and the Small Industries Development Bank of India (SIDBI) to establish an independent credit risk rating agency to expedite the processing of SSI loans and make the process more objective. The average loan size for SSI loans is approximately Rs. 400,000. Products and Services for Retail Customers General Our retail banking business provides financial products and services to our retail customers. We provide housing, retail trade, automobile, consumer, education and other personal loans and deposit services such as demand, savings and fixed deposits for our customers. We also provide supply chain financing to our retail trade customers. In addition, we provide fee based products and services such as cash management, remittance and collection services and safe deposit lockers to our customers and distribute products such as credit cards, mutual funds and non-life insurance issued and underwritten by other providers. 43 Retail Lending Activities Our total outstanding retail loans and advances were Rs. 99.7 billion as of September 30, 2004, representing 19.2% of our total outstanding loans. The following table classifies our outstanding retail loans by category of loan, as of March 31 and September 30, 2004: (In Rs. million) March 31, 2004 September 30, 2004 Number of Amount % of total Number of Amount % of total loans outstanding outstanding loans outstanding outstanding retail loans retail loans Housing 104,495 37,112.1 40.56% 118,391 42,278.7 42.43% Mortgage 7,507 4,154.7 4.54% 9,590 4,762.0 4.78% Traders 83,126 23,659.9 25.86% 103,084 25,835.1 25.92% Automobile 56,008 5,953.3 6.51% 56,874 5,752.7 5.77% Personal loans 90,927 3,985.3 4.36% 97,288 4,521.9 4.54% Consumer 61,883 2,094.4 2.29% 57,718 1,847.0 1.85% Pensioners 109,431 2,498.9 2.73% 120,961 2,687.3 2.70% Education 19,115 3,697.2 4.04% 22,447 4,304.7 4.32% Lease rentals 272 1,073.4 1.17% 350 2,034.2 2.04% Gold 25,423 423.6 0.46% 17,740 342.0 0.34% Doctors 1,781 768.8 0.84% 2,463 1,172.6 1.18% Other* N.A 6090.0 6.64% N.A 4,116.7 4.13% Total outstanding retail loans 559,968 91,511.6 100.00% 606,906 99,654.9 100.00% * Includes advances against national savings certificates, mutual fund units and other financial instruments, which have been classified as retail loans from April 1, 2004. The following is a description of our principal loan products: Housing Finance Our housing finance business involves giving long-term loans to individuals to finance the purchase, construction, repairs and renovation of a home. These loans are primarily secured by a charge over the property financed. These loans are extended for maturities up to 25 years and have an average size of approximately Rs. 400,000. We also offer a housing loan product in which, part of the loan is offered as an overdraft facility whereby an additional line of credit up to 20% of the housing loan is available to the customer after a period of five years. Mortgage Finance Through our mortgage finance business, we give loans to customers which can be used for personal or business needs against security over real property. These loans are extended for maturities up to six years and are given for amounts up to a maximum of Rs. 10 million. 44 Trade Finance Through our trade finance business, we provide financing to individuals, firms, co-operative societies and companies, typically in the form of revolving working capital facilities and term loans with maturities up to seven years. These loans are typically collateralized by a charge over movable or immovable assets. We provide supply-chain financing to this sector through, among others, our new product PNB Supply Chain Management. Such financing entails providing banking services such as overdraft facilities and bill discounting to suppliers and buyers. Automobile Finance We provide loans to finance the purchase of new and used automobiles, including two wheelers. Automobile loans are secured by a charge on the purchased automobile and typically also by a personal guarantee of a party other than the borrower. These loans are extended for a term of up to seven years for new vehicles and up to five years for used vehicles and have an average size of approximately Rs. 100,000. We also have non-exclusive arrangements with Hyundai Motors and Hero Honda Motors to provide financing for their products. Personal and Consumer Loans We provide personal loans for various purposes in amounts ranging from Rs. 10,000 to Rs. 300,000. These loans are typically made for a period of up to five years and are secured by personal guarantees from a party other than the borrower, including employers. In the case of consumer loans, the item purchased is hypothecated as security. We have introduced several new products in this area including PNB Joy, which is a loan of up to Rs. 200,000 for working couples. We also offer our Gold Loan Scheme, a product under which we make loans secured by gold and silver jewellery and ornaments. Loans to Pensioners We provide loans to pensioners in order to meet their personal requirements such as medical expenses and other needs. These loans are typically made for a term of two to four years and for amounts up to Rs. 100,000. Repayments on these loans are made from pension funds deposited in the pensioners’ accounts with us. Education Loans We provide loans for higher education which are typically for a term of five to seven years excluding the study period and are unsecured up to Rs. 750,000. Typically a parent acts as a co-borrower. The average size of these loans is approximately Rs. 200,000. Additionally, we offer our Sarvottam Shiksha product, which is an education loan with concessionary terms depending on the quality of the institution attended. Other Retail Products and Services Credit Cards We offer a credit card issued by HSBC on a co-branded basis. We earn a fee for sales of the card as well as transactions but do not currently have any balance sheet exposure on the cards. As of September 30, 2004 we had sold over 49,000 cards since the card was introduced in November 2000. Debit Cards We offer a debit card which bears the Maestro and Cirrus logos. Currently, our debit card has acceptance at approximately 45,000 merchants’ establishments and 4,200 ATMs across 70 cities in India. We had over 248,000 debit card customers as of September 30, 2004. Insurance We market the non-life insurance products of New India Assurance Co. Ltd. We earn commissions for these products but do not underwrite them. As of September 30, 2004 we had sold over 324,000 policies. Mutual Funds We distribute the mutual fund products of our joint venture Principal PNB Asset Management Co. Ltd. We earn fees for the distribution of the funds. 45 Depository Services We provide depository accounts and related services to individual retail customers in connection with the holding of debt and equity securities. We earn fees for maintaining the accounts as a depository participant of National Securities Depositary Ltd. as well as for the related services. Services to Non-resident Indians We provide personal financial services for NRIs. We have established a branch in Kabul and representative offices in other cities overseas in order to facilitate services being provided to NRIs. We offer foreign currency accounts to NRIs under our Foreign Currency Non-Resident Scheme and rupee accounts for NRIs under our Non-Resident External and Non-Resident Ordinary Schemes. We have introduced our Global Foreign Currency Scheme and Global Rupee Deposit Scheme, which offer benefits and concessions to NRIs and their relatives provided a minimum balance of Rs. 250,000 or US$5,000 is maintained in the account. We also offer various products for facilitating remittances from NRIs to India. We recently entered into an arrangement to facilitate money transfers through Western Union, which is a global leader in money transfer services. We have also entered into an agreement with Times Online Money Ltd., a Times of India group company, with a view to establishing an internet based international remittance service. In addition, we also provide housing loans to NRIs. Recent Retail Initiatives We plan to introduce the following products by March 2005: ● Online Bill Payment. This service is based on cost sharing arrangements with utility service providers, where we either charge the provider a fixed charge per bill or the provider maintains a balance with us before it uses the funds, resulting in low cost deposits with us. ● PNB Omni. Our PNB Omni product will provide concessional rates to customers who transfer all their loans from other lenders to us. ● PNB My Money. Our PNB My Money product will function as a personal overdraft facility and will allow customers to borrow from an overdraft account. Products and Services for Agriculture Customers General Agriculture contributes 22% to India’s GDP and supports approximately two-thirds of India’s population. In fiscal 2004, we surpassed the stated national goal that banks should provide at least 18% of their net bank credit (which is gross credit minus Foreign Currency Non-Resident Bank deposits) to this segment, for which we received an award from India’s Finance Minister. Our average credit growth rate in this segment has been 32.2% over the last four years. As of the last reporting Friday of September 2004, agricultural loans constituted 18.8% of our net bank credit. Currently, our agricultural operations are spread across India with a concentration in northern India, in the fertile areas of Punjab, Haryana, Uttar Pradesh and Rajasthan. These states in the aggregate accounted for 59.2% of our total agricultural loans as of the last reporting Friday of September, 2004. We intend to further increase our customer base in other areas in India. 46 The following table shows the value and share of our agricultural loan products as of the last reporting Friday of the months indicated: March 2004 September 2004 Geographic Number of Amount % of total Number of Amount % of total Distribution accounts outstanding outstanding accounts outstanding outstanding (in millions) (Rs. in billions) agricultural (in millions) (Rs. in billions) agricultural loans loans Northern: Punjab, 1.02 69.5 74.3% 1.09 78.1 72.5% Haryana, Rajasthan, Himachal Pradesh, Uttar Pradesh, Jammu and Kashmir, Uttranchal, Delhi Eastern: West 0.24 10.5 11.2% 0.26 11.6 10.8% Bengal, Bihar , Jharkhand, Northeastern states, Orissa Central: Madhya Pradesh, Chattisgarh 0.05 4.5 4.8% 0.06 5.0 4.6% Western: Gujarat, 0.01 6.1 6.5% 0.01 10.0 9.3% Maharashtra Southern: Tamil 0.06 3.0 3.2% 0.06 3.0 2.8% Nadu, Karnataka, Kerala , Andhra Pradesh, Pondicherry Total agriculture portfolio 1.38 93.6 100.0% 1.48 107.7 100.0% The agriculture sector is also advantageous in terms of risk and credit management, with a comparatively high number of smaller loans. Our gross non-performing assets in the agriculture segment constituted 4.37% of total agricultural advances as of September 30, 2004 as compared to gross NPAs of 7.65% as a percentage of gross advances. We have also been able to minimize our non performing assets through arrangements with different participants in the supply chain. For example, we enter into tripartite arrangements with sugar mills and groups of farmers in which the payments from the mill to the farmers are made through the farmers’ accounts with us. Since the sale proceeds are deposited with us, the risk of nonpayment is reduced. Recent regulatory developments have also improved the efficiency of the agricultural market and made it a more attractive lending sector. Through the National Agricultural Insurance Scheme, which is available in the states in which it has been adopted, farmers can purchase insurance coverage for their crops. This coverage is compulsory for farmers that are borrowers and the lender is a loss payee. For small farmers, 50% of the cost of this insurance is also subsidized by the central and state governments. Of the states listed in the table above Kerala, Maharashtra, Madhya Pradesh, Orissa, Himachal Pradesh, Gujarat, Uttranchal, Uttar Pradesh and Tamil Nadu have implemented this scheme as of the date of this Red Herring Prospectus. Agricultural Lending In the agriculture sector we offer direct financing to farmers for production and investment as well as indirect financing for infrastructure development and credit to suppliers of inputs. The following table shows the value and share of our agricultural 47 loan products as of the last reporting Friday of March and September 2004, classified by type: March 2004 September 2004 Number of Amount % of total Number of Amount % of total accounts outstanding outstanding accounts outstanding outstanding (in millions) (Rs. in billions) agricultural (in millions) (Rs. in billions) agricultural loans loans Direct financing: Production financing 0.70 26.7 28.6% 0.74 28.6 26.5% Investment financing 0.67 38.4 41.0% 0.73 43.9 40.8% Indirect financing 0.01 28.5 30.4% 0.01 35.2 32.7% Total agriculture 1.38 93.6 100.0% 1.48 107.7 100.0% portfolio Production Financing We provide short-term working capital for agricultural activities such as planting and crop production for a term of 12 to 18 months. This is typically secured by crops or other movable assets for amounts up to Rs. 50,000 and additionally by a mortgage over real property for amounts above Rs. 50,000. Investment Financing We provide financing for agricultural investments such as farm mechanization, irrigation, transport vehicles, development of dairy, poultry and fishery facilities and wasteland development. The term of such financing is typically five to seven years. This is typically secured by the assets being purchased and/or a mortgage of or charge on agricultural land or immovable property. Indirect Financing We provide indirect financing for infrastructure development and provide credit to dealers or distributors of inputs such as pesticides, seeds and fertilizers. These loans are typically secured by hypothecation of the assets financed and/or a mortgage of real property or a guarantee. Krishi Credit Cards In 1998, we introduced our “PNB Krishi card” which is designed to meet the production and consumption credit needs of farmers. The Krishi card is being extended to cover the investment needs of farmers. The card provides farmers a working capital facility up to certain limits. The farmer can withdraw money from any of our branches located in the district where the card issuing branch is located. It also allows them to make payments for supplies without carrying cash or having to process invoices. Additionally, farmers can use this card to meet short term domestic needs. Loans using the Krishi credit card up to Rs. 50,000 are secured by crops and assets financed by the loan, and advances over Rs. 50,000 are additionally secured by a mortgage over the farmer’s land. Krishi cards are typically set up for a period of three years but must be fully paid down at least once in 18 months (for long duration crops) and 12 months (for normal crops), except for the investment credit portion which is payable by installments. As of September 30, 2004, we had issued over 1.3 million Krishi credit cards. Agriculture and Community Support Efforts We intend to maintain our reputation as a premier agricultural financing institution in India. As part of our commitment to this sector, we provide a variety of programs to offer support and assistance to rural communities. We have established training centers for farmers to inform them of the latest technological developments in agriculture and related activities, educate them about adopting better production and marketing practices and provide them with information about the financing options and services available to them. We have also instituted support programs such as the PNB Farmers Welfare Trust which, in addition 48 to providing training to farmers, provides vocational training to women and youth, animal health treatment and other social services. Directed Lending The RBI guidelines require banks to lend at least at least 40% of their net bank credit to certain specified sectors called priority sectors. Priority sectors include the agricultural sector, SSIs, food and agri-based industries (with investments in plant and machinery up to Rs. 50 million), small business, self-employed and professional individuals, housing finance up to certain limits, and certain other sectors. Out of the 40%, banks are required to lend a minimum of 18% of their net bank credit to the agriculture sector. We are required to comply with the priority sector lending requirements on the last reporting Friday (alternate Fridays are designated by the RBI as “reporting Fridays”) of each fiscal year. Any shortfall in the amount required to be lent to priority sectors may be required to be deposited with Government sponsored development banks such as the National Bank for Agriculture and Rural Development and the Small Industries Development Bank of India. These deposits have a maturity of up to seven years but are relatively low yielding. We report our priority sector loans to the RBI on a quarterly basis. The loans reported are as of the last reporting Friday of the quarter. As of September 17, 2004, which was the last reporting Friday for the quarter ended September 30, 2004 our priority sector loans were Rs. 242.7 billion, constituting 47.7% of our net bank credit against the requirement of 40.0%. The agriculture sector constituted 44.4% of our priority sector credit and small scale industries (SSI) constituted approximately 23.6% of our priority sector credit as of the last reporting Friday of September, 2004. The following table presents data on our outstanding priority sector lending, including as a percentage of our total net bank credit, as of the last reporting Friday of the months indicated. (In Rs. billion, except for percentages) March 2003 March 2004 September 2004 Amount % of net Amount % of net Amount % of net bank credit (1) bank credit (1) bank credit (1) Agriculture credit 70.8 16.4% 93.6 18.5% 107.7 18.8% SSI credit 47.9 12.0% 57.0 12.3% 57.3 11.3% Other priority sector credit 67.8 17.0% 79.3 17.0% 77.7 15.3% Priority sector credit 186.6 46.7% 229.9 49.4% 242.7 47.7% Net bank credit 399.7 465.5 508.6 (1) Net bank credit is gross bank credit less Foreign Currency Non-resident (Bank) deposits. Our priority sector lending includes the agricultural and SSI lending described above, as well as certain of our loans to individuals for education and other purposes. In addition, priority sector lending includes housing finance up to Rs. 1.5 million. In addition to the foregoing, our priority sector activities also include micro credit. Our micro credit offerings provide financing to start-up businesses and other small scale entrepreneurs. The credit is primarily disbursed to Self-Help Groups (SHGs) which are composed mainly of women. Our micro credit disbursements up to September 30, 2004 were Rs. 1.2 billion to 24,115 SHGs. 49 Deposits Our deposit products include the following: ● demand deposits, which are non-interest bearing; ● savings deposits, which are deposits that accrue interest at a fixed rate set by the RBI (which is currently 3.5%) and upon which cheques can be drawn; and ● term deposits (which have interest rates determined by banks) including: ● recurring deposits, which are periodic deposits of a fixed amount over a fixed term that accrue interest at a fixed rate; and ● certificates of deposit. The following table sets forth the balances outstanding by type of deposit, as of the dates indicated: (Rs. in million, except for percentages) March 31, 2003 March 31, 2004 September 30, 2004 Balance % of total Balance % of total Balance % of total outstanding outstanding outstanding Demand deposits 98,882.8 13.0% 99,003.9 11.3% 97,482.1 10.3% Savings deposits 256,479.0 33.9% 304,226.1 34.6% 330,151.0 34.9% Term deposits 402,773.2 53.1% 475,934.0 54.1% 518,253.3 54.8% Total deposits 758,135.0 100.0% 879,164.0 100.0% 945,886.4 100.0% For a description of the RBI’s regulations applicable to deposits in India and required deposit insurance, see sections titled “Regulations and Policies — Regulations Relating to Deposits” and “Regulations and Policies — Deposit Insurance” on page 175. We take corporate deposits from large public sector corporations, government organizations, other banks and private sector companies. We take rupee or foreign currency denominated deposits and offer fixed and floating interest rates. In addition to our conventional deposit products, we offer a variety of special value-added products and services that complement our deposit accounts. “Prudent Sweep” and “Smart Roamer” accounts These products provide automatic transfer of balances beyond certain minimum amounts, from the demand deposit accounts to term deposit accounts, resulting in higher yields for the customer. Whenever there is a shortfall in the customer’s account, deposits are automatically transferred back from the term deposit account to meet the shortfall. We refer to this as a “sweep-in, sweep-out” feature. These products also enable the customer to access their account and the associated services from any of our over 690 branches (as of September 30, 2004) that are connected through our CBS system. PNB Auto-Sweep This is a current account for government organizations and PSUs with the “sweep-in, sweep-out” feature. PNB Flexible Rate Deposit Scheme This is a product which enables our customers to receive a flexible interest rate based on the yield on Government securities, which is reset every six months. 50 PNB Spectrum PNB Spectrum is a deposit account which has recently replaced the various term deposit products that were previously offered. It will provide a maturity option or income option to depositors opening accounts. Zero Balance Salary Account These special accounts have no minimum balance and offer other concessions to employees of companies that deposit their employees’ salaries directly with us. Cheque Truncation We have launched an intra-bank, inter-city project, which is intended to enable faster clearance of instruments at our three MICR (magnetic ink character recognition) centers in Chandigarh, Jalandhar and Ludhiana. This is a process in which cheque clearing beyond a certain stage in the cycle occurs on the basis of electronic data and images of the cheque, resulting in faster clearance. To legalize image based cheque payment, the Negotiable Instruments Act, 1881 was amended in December, 2002, paving the way for the clearing of cheques through this process. The project is expected to be extended to other MICR centers across India. Delivery Channels We deliver our products and services through a network of 4,034 branches, 452 extension counters and 467 ATMs in 160 cities throughout India, as of September 30, 2004. We also use ATMs, call centers and the Internet. Generally, our branches are organized into regions that are in turn organized into zones that report directly to our head office. Certain branches report directly to our head office as explained below. The table below shows our regional breakdown by branches, regions and zones as of September 30, 2004. (In Rs. billion) Branches CBS Regions Zones Deposits Advances branches (1) Northern: Punjab, Simla, 1,571 287 20 5 453.7 253.9 Northern Delhi, Rajasthan Eastern: West Bengal, Bihar, 776 46 11 3 105.4 46.8 Jharkhand, Northeastern states, Orissa Central: Central/West/East Uttar 1,121 170 15 6 237.4 77.5 Pradesh, Uttranchal, Madhya Pradesh, Chattisgarh Western: Mumbai, Gujarat 230 126 4 2 99.8 82.1 Southern: Chennai , Kerala 336 65 6 2 49.6 58.4 Total 4,034 694 56 18 945.9 518.7 (1) CBS Branches are those branches where we have implemented our Core Banking Solution, which interconnects our branches. Branches and Extension Counters Our branch network is the second largest in India, second only to the State Bank of India. We aim to rationalize our extensive branch network by merging branches which have become unnecessary and opening new branches in which meet our customers’ current needs. Our extension counters are small offices primarily within office buildings or on factory premises that provide commercial banking services. 364 of our branches are ISO 2000 certified. 51 We are in the process of centralizing our loan processing operations at regional processing centers. Using this “hub and spoke” organizational model, a portion of our loan activity will be centered in 25 centers, each covering five to ten branches. We believe this will facilitate faster delivery of credit, standardized decision making and centralized control during the life cycle of the loan. We have already established centers in 13 zones. We are currently modernizing our branch network with technology in order to increase efficiency and increase profits per employee. Because we have a large legacy branch network, our profits per employee are lower than many of our competitors, especially private banks that have relative new branch networks. We believe that improving productivity by implementing new technology and upgrading our branches is an area where we have significant room to improve, and we expect to realize future improvements in total profit as a result of these efforts. Following is a table of our business (advances plus deposits) per employee and average gross profit per branch for the periods indicated. (Rs. in thousands, except for employees per branch) Fiscal Year First six months of fiscal year 2002 2003 2004 2004 2005 Business per employee 16,776 19,564 22,822 20,371 24,855 Business per branch 251,640 285,830 333,886 296,920 361,870 Gross profit per branch 3,821 5,740 7,759 8,560* 8,100* Employees per branch 15.00 14.61 14.63 14.56 14.56 * Figures provided on an annualized basis. We also have specialized branches targeting various segments of our customers. We have specialized corporate branches focused on the needs of small, medium and large sized corporations. We have two branches specializing in our large sized corporate customers which report directly to our head office. We currently have five branches targeting trade finance and plan to set up others specializing in SSI, retail finance, housing finance, high value customers, families and senior citizens. We also have 126 “signature” branches which offer personalized service and advanced technology combined with a welcoming décor. As a part of its branch licensing conditions, the RBI has stipulated that at least 25% of our branches must be located in semi- urban and rural areas. As of September 30, 2004, 2,728, or 67.6% of our branches were located in semi-urban and rural areas. Interconnectivity of Branches We have implemented our Core Banking Solution, in which our branches are interconnected electronically, in 694 branches which represent approximately seven million customers and 46.7% of deposits and advances by amount, as of September 30, 2004. We have a centralized server in Delhi to connect these branches and a back-up server in Mumbai. This allows customers to access their accounts and obtain services from any connected branch as if they were in their primary branch. This provides greater mobility and convenience for our customers and we believe will enhance customer loyalty to us. We believe that this will enable us to provide a greater range and volume of value added services to our customers. We plan to extend this interconnectivity to between 1,500 and 2,000 branches by March 31, 2006. We have set up a disaster recovery site at Mumbai, India. The site facilitates storage and back-up of data from the CBS branches so that if the data is lost or corrupted, it may be restored by using data at the site. This ensures the continuation of our critical business operations in the event of unforeseen circumstances. With the implementation of CBS, we have become a leader in IT implementation among public sector banks in India. Our efforts in this direction have been recognized by organizations such as NASSCOM (Best IT User 2004 Award) and CIOL Dataquest (Enterprise Connect Award 2004). We have achieved our target of computerizing all of our branches. 52 International Branches We have recently opened a branch in Kabul, Afghanistan and representative offices in London (UK), Almaty (Kazakhstan) and Shanghai (China). These international locations service the needs of Indian communities in other countries and also facilitate import/export trade. ATMs We offer our customers an extended network of ATMs with 467 ATMs in 160 cities as of September 30, 2004. We have recently entered into an ATM sharing arrangement called “MITR” with four other banks, namely UTI Bank, Indian Bank, Oriental Bank of Commerce and the erstwhile Global Trust Bank. Karur Vyasa Bank joined MITR on September 21, 2004. This arrangement allows our customers to access over 2,500 ATMs. Similarly, customers of these other banks can also access their bank accounts using our ATM network. We receive fees for this service. In view of the diversity of regional languages used in various parts of India, some of our ATMs offer bilingual screens. Internet We believe that Internet access and information is key to satisfying the needs of certain customer segments. We maintain a website at www.pnbindia.com, offering general information on our products and services. Our Internet banking service allows customers to access all their account-related information and also enables them to transfer funds from one account to another account with us, using our website. This facility is available to both individual and corporate customers of our interconnected branches. Call Center We provide customer support services through our call center in Gurgaon , near Delhi. The call centre functions 24 hours a day, seven days a week, and offers a self-service option to customers for automated phone banking. In addition, customer service officers offer personalized services for banking customers, credit card holders, bond holders and loan product customers. As part of our cross-selling initiatives, the call center is also used to market our various products. As of September 30, 2004, our call center took an average of 973 calls per day. Treasury Through our treasury operations, we seek to manage our balance sheet, including the maintenance of required regulatory reserves, and earn profits from our trading portfolio. Our treasury operations also include a range of products and services for corporate and commercial customers, such as forward contracts and interest rate and currency swaps, and foreign exchange products and services. Income from our treasury and trading operations is subject to volatility, as discussed in the section titled “Risk Factors” on page x. General Under the RBI’s statutory liquidity ratio (SLR) requirement, we are required to maintain an amount equal to at least 25.0% of our net demand and time liabilities in approved securities, such as Government of India securities, state government securities and other approved securities. As of September 30, 2004, 38.2% of our net demand and time liabilities consisted of Government and other approved securities. Under the RBI’s cash reserve ratio requirements, we are required to maintain a minimum of 5% of our net demand and time liabilities in a current account with the RBI. As of September 30, 2004, 5.1% of our net demand and time liabilities were maintained with the RBI. The RBI pays no interest on these cash reserves up to 3.0% of the net demand and time liabilities and pays interest at 3.5% per annum on the remaining eligible balance. For further discussion of these regulatory requirements, see the section titled “Regulations and Policies - Legal Reserve Requirements” on page 175. Our treasury undertakes liquidity management by seeking to maintain an optimum level of liquidity while complying with the cash reserve and statutory liquidity ratios. We maintain the statutory liquidity ratio through a portfolio of Government of India securities that we actively manage to optimize the yield and benefit from price movements. Our Board of Directors approves our investment and asset liability management policies. 53 The following table sets forth, as of the dates indicated, the allocation of our investment portfolio. March 31, 2003 March 31, 2004 September 30, 2004 Securities In Rs. % In Rs. % In Rs. % millions millions millions SLR Government and approved securities 277,530 81.6% 354,183 84.1% 386,691 84.0% Non-SLR Bonds and debentures 54,584 16.0% 57,663 13.7% 57,319 12.4% Shares 2,701 0.8% 2,667 0.6% 3,030 0.7% Subsidiaries and joint ventures 2,229 0.7% 2,090 0.5% 3,100 0.7% Commercial papers, mutual funds and others 3,256 0.9% 4,652 1.1% 10,046 2.2% Total 340,300 100.0% 421,255 100.0% 460,186 100.0% The following table presents our investments as a percentage of our total assets and our investment income as a percentage of our total income as of or for the dates indicated. (Rs. in billions, except percentages) March 31, 2003 March 31, 2004 September 30, 2004 Average total interest earning assets 720.9 856.9 1,002.2 of which, Average investments 317.8 403.4 480.0 Average investments as a percentage share of average total interest earning assets 44.1% 47.1% 47.9% (Rs. in billions, except percentages) Fiscal year First six months of fiscal year 2003 2004 2004 2005 Total income 87.4 96.5 48.7 51.9 Income from investments 33.0 36.8 17.8 19.7 Net profit from sale of investments 6.7 12.4 7.0 2.8 Total treasury income* 39.7 49.2 24.8 22.5 Treasury income as a percentage of total income 45.4% 51.0% 50.9% 43.4% 54 * The above does not include this income earned on the following: 1. income earned from call money and short term investments; 2. income from derivatives; and 3. dividend earned on investments in subsidiaries and joint ventures. Foreign Exchange Activities We provide customer specific products and services and risk hedging solutions in several currencies to meet the trade and service-related requirements of our corporate and commercial clients. The products and services offered include: ● foreign exchange at spot rates for the conversion of foreign currencies without any value restrictions; ● forward foreign exchange contracts for hedging future receivables and payables, without any value restriction; and ● foreign exchange and interest rate derivatives for hedging long-term exposures. We commenced our derivative operations in fiscal 2003. We offer to our customers forward foreign currency products, single currency interest rate swaps and cross-currency interest rate swaps. We have hedged a part of our fixed liabilities with floating rate swaps. The primary purposes of our trading in interest rate derivatives is hedge our position but we also trade in interest rate derivatives to take advantage of volatility in the market. In fiscal 2004 and the first half of fiscal 2005, our net income from derivatives operations was Rs. 148 million and Rs. 120 million, respectively. Ratings Information Our Tier 2 bonds are rated LAAA by ICRA Limited. Our term deposits are rated MAAA by ICRA Limited. Our Subsidiaries and Affiliates We provide additional products and services through our subsidiaries and affiliates. PNB Gilts Ltd We owned 74.1% of PNB Gilts Ltd. as of September 30, 2004, which was formed in 1996. PNB Gilts Ltd. is licensed as a primary dealer in the government securities market. PNB Gilts. Ltd deals in Government securities, treasury bills, bond and other money market instruments. PNB Gilts Ltd. generated income of Rs. 2,379 million in fiscal 2004 and a loss before operating expenses of Rs. 512 million in the first half of fiscal 2005. It generated a net profit of Rs. 1,070 million in fiscal 2004 and had a loss of Rs. 871 million in the first half of fiscal 2005, which was primarily due to trading losses incurred by it as a result of increasing interest rates during the period. PNB Housing Finance Ltd. PNB Housing Finance Ltd., our wholly owned subsidiary formed in 1988, is engaged in providing housing loans for the purchase, construction and renovation of dwellings. The total income of PNB Housing Finance Ltd. was Rs. 904 million in fiscal 2004 and 430 million in the first half of fiscal 2005. Its net profit was Rs. 155 million in fiscal 2004 and Rs. 78 million in the first half of fiscal 2005. Asset Reconstruction Companies Legislation in 2002 allowed the creation of asset reconstructions companies (ARCs), which are allowed to buy non-performing assets from banks at a discount from book value. These entities earn returns by aggregating non-performing assets and recovering value from them, which is possible without the use of litigation. Additionally, ARCs use these assets to sell asset backed securities through special purpose vehicles. We own a stake in two such companies. We own 26% of Assets Care Enterprise Ltd., and recently acquired 10% of Assets Reconstruction Company of India Ltd. Assets Reconstruction Company of India Ltd. has begun asset acquisition activities, while Assets Care Enterprise Ltd. has not yet started asset acquisition activities. Regional Rural Banks We have promoted 19 regional rural banks, with 1,282 branches spread over seven states of India as of September 30, 2004. We hold 35% of the equity share capital of the regional rural banks and the Government of India and state governments own 50% 55 and 15%, respectively. As of September 30, 2004 the deposits of these banks were Rs. 54.5 billion and advances were Rs. 22.4 billion. The combined net profit of these regional rural banks improved by 16.1% from Rs. 1.3 billion in fiscal 2003 to Rs. 1.5 billion in fiscal 2004 and was Rs. 0.6 billion in the first six months of 2005. Gross NPAs for these banks amounted to 7.25% of their total advances as of September 30, 2004. Our board has recently approved the amalgamation of some of our regional rural banks in certain states with a view to strengthening them and improving their efficiency. Risk Management We believe that the three principal risks to our business are credit, market and operational risk. We have set up a Risk Management Committee of our Board of Directors which manages these principal risks. The Risk Management Committee supervises the implementation of risk management policies and is assisted by three committees of our senior executives: the Credit Risk Management Committee (for credit risk), the Asset Liability Management Committee (for market risk) and the Operational Risk Management Committee (for operational risk). Credit Risk Credit risk is defined as the possibility of losses associated with the diminution in the credit quality of the borrower or counter party or failure on its part to meet its obligations in accordance with the agreed terms. To monitor and manage credit risk, we have set up a Risk Management Division at our head office and Credit Risk Management Department at each zonal office. Credit risk is managed through prudent selection of borrowers, delegation of appropriate lending powers and by setting up various prudential limits. The management of credit risk is based on clearly defined responsibilities and procedures approved by our Credit Risk Management Committee. We seek to constantly upgrade the risk evaluation skills of our employees at every level through specialized training programs. For data on our credit risk exposure, see the section titled “Selected Statistical Information” on page 72. Credit Approval and Monitoring The principal means of managing credit risk is the credit approval process. We have developed various credit risk rating models. We apply our risk rating criteria to all individual advances over Rs. 2 million (prior to September 2004 we applied these criteria to advances over Rs. 5 million). Every eligible account is assigned a credit rating. We have also developed industry ratings which we use as an element of our rating of individual borrowers. As of September 30, 2004, our individually rated loans amounted in the aggregate to Rs. 249.4 billion or 48.9% of our total outstanding loans and 49.7% of our total standard advances (total loans net of NPAs). We have seven ratings categories for our loans which we categorize as investment grade, marginally acceptable grade and sub- investment grade. The table below shows the distribution of our loans as of September 30, 2004 by rating and grade. Rating Amount Percentage of Grade Percentage of Outstanding total total (Rs. in millions) outstanding outstanding AAA 6,568 2.6% Investment Grade 68.8% AA 27,606 11.1% A 65,163 26.1% BB 72,400 29.0% B 40,572 16.3% Marginally Acceptable Grade 16.3% C 27,059 10.9% Sub-Investment Grade 14.9% D 10,073 4.0% Total 249,441 100.0% 100.0% All rated credits are discussed at our head office and each zonal office by a Credit Committee composed of officers from the Risk Management and Credit Operations divisions. The final approval is granted by a competent authority at different levels of the 56 organization depending on the size of the credit. For example, any loan above Rs. 300 million must be approved by the Management Committee of our Board of Directors. We have 332 loans currently outstanding in excess of Rs. 300 million as of September 30, 2004. For certain good borrowers, we permit certain categories of officials to extend ad hoc facilities in excess of specified limits in exceptional circumstances in order to meet emergent requirements. Our credit policies are based primarily on statistical analyses of risks with respect to different products and types of customers. We have high volumes of relatively homogeneous, small value transactions in the retail and agricultural areas. We monitor our own and industry experience to determine and periodically revise product terms and desired customer profiles. We then verify that an individual customer meets our lending criteria. A large number of our loans are also secured by collateral or supported by guarantees. Our trade and commercial loans are typically secured by a charge over inventory or receivables. Longer term loans are usually secured by a charge over fixed assets. Our larger retail loans are typically secured by collateral or guaranteed. Our retail loans are generally either secured, made against delivery of post-dated cheques or debited to an account maintained with the bank in which the debtor’s employer directly deposits their salary. In India bouncing of cheques is a criminal offence. In the case of most automobile loans as well as unsecured personal loans, we require that the borrower provide post-dated cheques for all payments on the loan at the time the loan is made. Automobile loans and consumer loans are both secured loans. We will generally lend up to 80% to 90% of the value of the automobile in case of automobile loans and 90% to 95% in the case of two wheeler loans. Our housing loans are secured by mortgages. Although we take collateral, we may sometimes not be able to realize its full value in a default situation. See the section titled “Risk Factors-We may experience delays in enforcing our collateral when borrowers default on their obligations to us, which may result in failure to recover the expected value of the collateral” on page xv. We aim to improve recovery by implementing our rule based lending system through out our branches. This system analyzes available data to profile areas and categories which we use to make our lending decisions more efficient. We are using software already implemented in our 170 branches which have bulk business in order to appraise and monitor accounts. The accounts are further reviewed and monitored through our Loan Review Mechanism. We rely on our Preventive Monitoring System which scores accounts based on such factors as financial performance and conduct of accounts on a quarterly basis with a rule based system. Additionally, local task forces monitor accounts that are in arrears or have low credit ratings. Our Credit Audit and Review Section also reviews all accounts above Rs. 35 million and accounts above Rs. 10 million identified as irregular or weak. Prudential Limits We have established our own prudential limits for funded exposure to individual industries and for borrowers and groups of borrowers, regardless of whether the exposure is in the form of loans or non-funded exposures. These limits are as follows: ● We have a limit of Rs. 250 million and Rs. 750 million for proprietorship and partnership concerns, respectively. ● Our substantial exposure (the cumulative exposure to those entities that have limits in excess of 10% of our capital funds) is limited to 400% of capital funds, which consists of Tier 1 and Tier 2 capital. ● Our credit exposure to any particular industry is limited to 10% of our total credit. This exposure includes accounts with total limits (fund based and non-fund based) or total outstandings (fund based and non-fund based) of Rs. 10 million and above. The exposure is then arrived at by adding the fund based outstanding of these accounts. As of September 30, 2004, our largest exposure to a single industry was infrastructure, which was 9.9% of our total fund based exposure to accounts of Rs. 10 million and above. ● We have lower limits for certain especially sensitive sectors such as the film industry, real estate, non-banking financial companies and capital markets. Our exposure limits are typically more conservative than those required by the RBI. Under the RBI guidelines, our exposure to individual borrowers must not exceed 15.0% of capital funds. Exposure to individual borrowers may exceed the exposure norm of 15.0% of capital funds by an additional 5.0% (i.e. up to 20.0%) provided the additional exposure is on account of infrastructure financing. Under the RBI guidelines exposure to a group of companies under the same management control must not exceed 40.0% of capital funds unless the exposure is in respect of an infrastructure project in which case the ceiling is 50%. Our prudential limits require the exposure to an individual borrower may not exceed 12.5% of capital funds (or 15% for 57 infrastructure) and exposure to a group of companies under the same management may not exceed 30% of capital funds (or 40% for infrastructure). Pursuant to the RBI guidelines, exposure for funded facilities is calculated as the total approved limit or the outstanding funded amount, whichever is higher. Exposure for non-funded facilities is calculated as 100.0% of the approved amount or the outstanding non-funded amount, whichever is higher (until year-end fiscal 2003 this amount was 50.0%). We are currently in compliance with these limits. As of September 30, 2004, we had exposure in excess of the 15% ceiling in two accounts, in which the exposures were 17.8% and 16.5% of our capital funds, for which we obtained RBI approval. On September 30, 2004, our largest exposure to a single borrower was 17.8% of our capital funds, and our largest group exposure was 28.6% of our capital funds; all these borrowers are currently performing according to the terms of our contracts with them. The RBI has advised that banks may, in exceptional circumstances, with the approval of their Boards, consider enhancement of the exposure to a borrower by a further 5% of the capital funds. Market Risk Market risk refers to potential losses arising from volatility in interest rates, foreign exchange rates, equity prices and commodity prices. Market risk arises with respect to all market risk sensitive financial instruments, including securities, foreign exchange contracts, equity and derivative instruments, as well as from balance sheet or structural positions. The objective of market risk management is to avoid excessive exposure of our earnings and equity to loss and to reduce our exposure to the volatility inherent in financial instruments. We consider interest rate risk to be potentially our most significant risk. A committee of our senior executives, the Asset-Liability Management Committee, is charged with the responsibility of managing liquidity and market risk. In the case of market risk, our exposure under commodities, equities and foreign exchange is a small proportion of the total balance sheet and the impact of such risks is further minimized due to adherence to the RBI’s stipulated norms relating to open positions and exposure ceilings. Asset Liability Management We generally fund our core customer assets, consisting of loans and credit substitutes, with our core customer liabilities, consisting principally of deposits. We also borrow in the short-term interbank market, though we are a lender most of the time. We use the majority of our funds to make loans or purchase securities. We maintain a substantial portfolio of liquid high-quality Indian government securities. While generally this market provides an adequate amount of liquidity, the interest rates at which funds are available can sometimes be volatile. We prepare regular maturity gap analyses to review our liquidity position, and must submit a monthly analysis to the RBI. The RBI imposes a limit on the negative mismatch during 1-14 day and 15-28 day periods, which should not exceed 20% of the outflow in the respective periods. As advised by the RBI, we have also established cumulative gap limits for all maturity buckets. We measure our exposure to fluctuations in interest rates primarily by way of a gap analysis on a monthly basis. We classify all rate sensitive assets and liabilities into various time period categories according to contracted residual maturities or anticipated re-pricing dates, whichever is earlier. The difference in the amount of assets and liabilities maturing or being re-priced in any time period category gives us an indication of the extent to which we are exposed to the risk of potential changes in the margins on new or re-priced assets and liabilities. Liquidity Risk The purpose of liquidity management is to ensure sufficient cash flow to meet all financial commitments and to capitalize on opportunities for business expansion. This includes our ability to meet deposit withdrawals either on demand or at contractual maturity, to repay borrowings as they mature and to make new loans and investments as opportunities arise. Our treasury group is responsible for ensuring that we have adequate liquidity, ensuring that our funding mix is appropriate so as to avoid maturity mismatches and to prevent price and reinvestment rate risk in case of a maturity gap, and monitoring local markets for the adequacy of funding liquidity. Price Risk Price risk is the risk arising from price fluctuations due to market factors, such as changes in interest rates and exchange rates. We consider interest rate risk to be potentially the most significant and for this purpose our Board has set limits on the maximum permissible impact on the net interest income allowed due to adverse interest rate changes during any one year period. As 58 stipulated by the RBI, we maintain an Investment Fluctuation Reserve to serve as a buffer against adverse movement in interest rates and to have sufficient capital to meet capital adequacy standards of minimum capital of 9% against total risk weighted assets. We have also begun to manage our risk by hedging through derivative transactions. We have also set prudential limits to manage our exchange risk including our daylight limit of US$44.3 million, our aggregate gap limit of US$1 billion and our overnight open position limit of Rs. 150 million. Operational Risk Operational risks are risks arising from matters such as non-adherence to systems and procedures or fraud resulting in financial loss. We have established systems and procedures to reduce operational risk. Compliance with our guidelines is monitored through internal control and a comprehensive audit system. A committee of our senior executives, the Operational Risk Management Committee, has been set up to oversee and monitor implementation of our Operational Risk Management Guidelines. Operational risks at the branch level have been identified and classified as control risks and business risks. Our risk profile format has been developed for preparing the overall operational risk profiles of our branches and to assess their level of risk. After assessing the overall risk of a branch, we take measures to minimize risk. Based on the level and direction of risk, a branch specific action plan is prepared with specific deadlines for mitigating and controlling the risk. To ensure compliance or rectification, compliance audits are conducted in selected branches. Senior officers also assess and evaluate the mitigating measures taken by the branch during their visits. We target high risk areas and branches for more frequent audit. Pursuant to RBI guidelines, some activities are required to be audited continuously. More than 60% of our business, measured by transaction volume, is subject to concurrent auditing, including foreign exchange, derivatives, equities, securities transactions, depositary services, retail liability operations, reversals to the profit and loss account and monitoring of inter-branch routing accounts. All other lines of business, our information technology department, and branches are audited on a set schedule, which is usually annual. Our information technology and software is also subject to audit review and certification, including application software and system controls. We are also subject to inspections conducted by the RBI under the Banking Regulation Act. The RBI has adopted the global practice of subjecting banks to examination on the basis of the CAMELS model, a model that assigns confidential ratings to banks based on their capital adequacy, asset quality, management, earnings, liquidity and systems. Basel II Requirements The RBI is adopting the requirements of Basel II, the international capital adequacy framework for banks. We are in the process of moving towards meeting these guidelines. These requirements will affect our management of all three principal categories of risk. In particular, Basel II will introduce minimum capital requirements for market risk and operational risk in addition to the previous requirement of minimum capital only for credit risk. Although we are on schedule to meet the requirements to be implemented, the adoption of these new rules is still an evolving process. We are regularly in contact with the RBI concerning the process of implementation of the Basel II guidelines and have been nominated by the RBI to be a member of the Steering Committee for the implementation of Basel II. Competition We face competition in all of our principal lines of business. Our primary competitors are other large public sector banks, new private sector banks and foreign banks. Commercial Banking Our principal competitors in wholesale banking are public and new private sector banks as well as foreign banks. The large public sector banks have traditionally been the market leaders in commercial lending. Foreign banks have focused primarily on serving the needs of multinational companies and Indian corporations with cross-border financing requirements including trade, transactional and foreign exchange services, while the large public sector banks have extensive branch networks and large local currency funding capabilities. 59 Retail Banking In retail banking, our principal competitors are the large public sector banks, as well as existing and new private sector banks and foreign banks in the case of retail loan products. The other public sector banks have large deposit bases and large branch networks, including the State Bank of India which has 13,593 branches. Private sector and foreign banks compete principally by offering a wider range of products as well as greater technological sophistication in some cases. Foreign banks, while having a small market penetration overall, have a significant presence among non-resident Indians and also compete for non-branch based products such as auto loans and credit cards. In particular, we face significant competition primarily from private sector banks and to a lesser degree from other public sector banks, in the housing, auto and personal loan segments. In mutual fund sales and other investment related products, our principal competitors are brokers, foreign banks and new private sector banks. Agriculture and Priority Segments In the agriculture and priority segments, our principal competitors are the large public sector banks, which have large branch networks and traditionally focused on these areas. Treasury In our treasury services for corporate and commercial clients, we compete principally with foreign banks in foreign exchange and derivatives trading, as well as the State Bank of India and other public sector and private banks in the foreign exchange and money markets business. Employees As of September 30, 2004 we had 58,747 employees. Our staff strength comprises 17,759 officers, 29,806 clerks and 11,182 sub-staff. Following is a table of our (unconsolidated) total number of employees as of the dates indicated: March 31, September 30, 2002 2003 2004 2004 Professional/technical employees 408 530 882 877 Other employees 57,451 58,451 57,957 57,870 Total 57,859 58,981 58,839 58,747 We do not have a stock incentive plan or discretionary bonus compensation. Bonus is paid in accordance with the Payment of Bonus Act for employees of basic salary and allowances of up to Rs. 3,500 per month. We also have incentive awards generally given to all employees of a branch for performance by the branch in various categories, such as having the lowest NPA levels. Over 95% of our employees are represented by unions or officers’ associations. We consider employee relations to be good. Negotiations to set salary and benefit standards for employees takes place at the industry level through the Indian Banks Association, which is an independent body. There is collective bargaining on the industry level. We provide a number of benefits for our employees, including canteen facilities, scholarships, part-time medical consultants, ex-gratia payments and partial adjustments of housing loans in case of death of employees while in service. We have ten training centers in India which conduct training programs to meet the changing skill requirements of employees in the core areas of treasury, risk management, retail based products and fee based products. We also have an IT training center and 20 Core Banking Solution laboratories for upgrading the IT skills of our employees. Our training programs include orientation sessions for new employees and management development programs for mid-level and senior executives. Our training centers regularly offer courses conducted by faculty, both national and international, drawn from industry, academia and from our own employees. Training programs are also conducted for developing functional as well as managerial skills. We also send our employees to various specialized training programs held in India and abroad. 60 Properties Our head office is located at 7, Bhikhaiji Cama Place, New Delhi 110 066, India. We have a principal network of 4,034 branches, 467 ATMs and 452 extension counters as of September 30, 2004. We own some branches directly, but most of our branches are leased. In addition to these, we own or lease our 18 zonal offices, 56 regional offices, three head office properties and a data centre in New Delhi. We also own residential apartments for the use of our employees. 61 HISTORY AND CERTAIN CORPORATE MATTERS History and Major Events We were incorporated under the Indian Companies Act, 1882 (Act VI of 1882) in 1894 as Punjab National Bank Limited and commenced operations on April 12, 1895 from Lahore . Upon nationalisation in 1969, we were renamed Punjab National Bank. 1904-13 ● The Bank expanded from Punjab to Karachi and Peshawar in 1904. ● In 1908, the Bank began its first dealings abroad by asking its Bombay branch to open an account in London. 1914-1921 ● In 1914, the Bank made a public issue of 5,000 shares. ● In 1919, the Bank took steps to establish an agency at London and an agency account with the London City & Midland Bank Ltd. 1922-1939 ● In 1926, the Bank was admitted to the clearing house of Calcutta. ● In 1928, Bhagat Ishwar Das became the first ‘Chairman Emeritus’ of the Bank. ● In 1929, a system of continuous audit was introduced to prevent possibility of fraud and the inspection of staff was strengthened. 1940-49 ● In January 1940 the Bank made its first acquisition of Bhagwandas Bank Limited. ● On June 29, 1947 the registered office of the Bank was shifted from Lahore to New Delhi. ● In 1949, the Punjab National Bank Workmen’s Union came into existence. 1950- till date ● In 1952, Bharat Bank merged with us. ● In 1956, Indo Commercial Bank merged with us. ● The first overseas branch of the Bank was opened in London in 1978 and closed in 1986 under instructions from the RBI. ● In 1961, Universal Bank of India merged with the Bank. ● In 1969, 14 private banks including our Bank were nationalised. ● In 1986, Hindustan Commercial Bank merged with the Bank. ● In 1993, New Bank of India merged with the Bank, which was the first ever merger of a nationalised bank with another. ● In 2002, we had an initial public offering of shares of the Bank which resulted in the reduction of the government’s shareholding in the Bank. ● In 2003, the erstwhile Nedungadi Bank, a Kerala based private bank, was amalgamated with the Bank. ● In 2003, PNB Capital Services Limited merged with the Bank. ● The number of branches we operated grew from 619 at the time of nationalisation in 1969 to reach 4,224 by September 2004. Awards and Recognitions ● In 2000, the ‘Bank of the Year Award’ given by the Banker’s Magazine of Financial Times, London. ● In 2000-2001, second prize in the GoI’s Indira Gandhi Rajbhasha Shield Pratiyogita. ● In 2001, 2002 and 2003, ‘Niryat Bandhu Gold Trophy Award’ by Federation of Indian Exporters Organisation for excellence in export performance. 62 ● In 2002-03, 2003-04 and 2004-05, second for its contribution to lending in the small scale industries sector for the years 2001-02, 2002-03, 2003-04. ● In 2002-03 and 2003-04, first prize from the RBI for the “PNB Staff Journal”, the in house journal of our Company. ● In 2002, 2003 and 2004, National Award for Excellence ranking second for our contribution in lending to the small scale industries sector. ● In 2002, Prime Ministers recognition for outstanding contribution to khadi, village and cottage industries. ● In 2002, ‘Best Bank Award’ for excellence in banking technology by Institute of Development and Research in Banking Technology (IDRBT). ● In 2002, ‘Best Bank Award’ for excellence in promotion of Kisan Credit Card Scheme. ● In 2002, Golden Peacock Award for excellence in corporate governance for the year 2001-02, by the Institute of Directors. ● In 2003, the one of our branches was awarded the ‘Best Branch’ among public sector banks by IDRBT. ● In 2002-2003, first prize in excellence in lending to the rural employment generation programme of khadi and village industries by the GoI for the year 2001-02. ● In 2004, Golden Peacock National Training Award by Institute of Directors. ● In 2004, Best IT User Award in Banking & Financial Services Industry by National Association of Software and Service Companies. ● In 2004, Enterprise Connect Award from CIOL Dataguest. Main Objects of Constitutional Documents of the Bank Section 3(5) of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 states as follows: “Every corresponding new bank shall carry on and transact the business of banking as defined in clause (b) of Section 5 of the Banking Regulation Act, 1949 (10 of 1949) and may engage in one or more of the other forms of business specified in sub- section (1) of Section 6 of that Act.” Section 5(b) of the Banking Regulation Act reads as follows: “ ‘banking’ means the accepting, for the purpose of lending or investment, of deposits of money from the public, repayable on demand or otherwise, and withdrawable by cheque, draft, order or otherwise.” Section 6(1) of the Banking Regulation Act reads as follows: “Form and business in which banking companies may engage (1) In addition to the business of banking, a banking company may engage in any one or more of the following forms of business, namely— (a) the borrowing, raising, or taking up of money; the lending or advancing of money either upon or without security; the drawing, making, accepting, discounting, buying, selling, collecting and dealing in bills of exchange, hoondees, promissory notes, coupons, drafts, bills of lading, railway receipts, warrants, debentures, certificates, scrips and other instruments and securities whether transferable or negotiable or not; the granting and issuing of letters of credit, traveller’s cheques and circular notes; the buying, selling and dealing in bullion and specie; the buying and selling of foreign exchange including foreign bank notes; the acquiring, holding, issuing on commission, underwriting and dealing in stock, funds, shares, debentures, debenture stock, bonds, obligations, securities and investments of all kinds; the purchasing and selling of bonds, scrips or other forms of securities on behalf of constituents or others, the negotiating of loans and advances; the receiving of all kinds of bonds, scrips or valuables on deposit or for safe custody or otherwise; the providing of safe deposit vaults; the collecting and transmitting of money and securities; (b) acting as agents for any Government or local authority or any other person or persons; the carrying on of agency business of any description including the clearing and forwarding of goods, giving of receipts and discharges and otherwise acting as an attorney on behalf of customers, but excluding the business of a managing agent or secretary and treasurer of a company; 63 (c) contracting for public and private loans and negotiating and issuing the same; (d) the effecting, insuring, guaranteeing, underwriting, participating in managing and carrying out of any issue, public or private, of State, municipal or other loans or of shares, stock, debentures, or debenture stock of any company, corporation or association and the lending of money for the purpose of any such issue; (e) carrying on and transacting every kind of guarantee and indemnity business; (f) managing, selling and realising any property which may come into the possession of the company in satisfaction or part satisfaction of any of its claims; (g) acquiring and holding and generally dealing with any property or any right, title or interest in any such property which may form the security or part of the security for any loans or advances or which may be connected with any such security; (h) undertaking and executing trusts; (i) undertaking the administration of estates as executor, trustee or otherwise; (j) establishing and supporting or aiding in the establishment and support of associations, institutions, funds, trusts and conveniences calculated to benefit employees or ex-employees of the company or the dependents or connections of such persons; granting pensions and allowances and making payments towards insurance; subscribing to or guaranteeing moneys for charitable or benevolent objects or for any exhibition or for any public, general or useful object; (k) the acquisition, construction, maintenance and alteration of any building or works necessary or convenient for the purposes of the company; (l) selling, improving, managing, developing, exchanging, leasing, mortgaging, disposing of or turning into account or otherwise dealing with all or any part of the property and rights of the company; (m) acquiring and undertaking the whole or any part of the business of any person or company, when such business is of a nature enumerated or described in this sub- section; (n) doing all such other things as are incidental or conducive to the promotion or advancement of the business of the company; (o) any other form of business which the Central Government may, by notification in the Official Gazette, specify as a form of business in which it is lawful for a banking company to engage.” Changes in Memorandum and Articles of Association We do not have any articles or memorandum of association as we are a “corresponding new bank” under the provisions of the Bank Acquisition Act. 64 Stock Market Data Stock Market Data of Our Equity Shares Our Equity Shares are listed on BSE and NSE. The following table sets forth, for the periods indicated, the high and low of daily closing prices of our Equity Shares on the NSE. (In Rs.) Period High Low Average(1) Fiscal year 2003 105.95 37.15 56.10 Fiscal year 2004 349.35 98.30 189.77 April 2004 387.35 333.25 353.89 May 2004 376.35 228.20 304.35 June 2004 297.75 242.05 264.65 July 2004 288.40 237.95 266.12 August 2004 275.65 243.20 262.02 September 2004 277.15 254.65 269.93 October 2004 268.30 241.95 254.59 November 2004 348.85 246.70 286.21 December 2004 405.60 351.15 371.59 January 2005 434.90 352.10 381.89 (1) Average of the daily closing price of our Equity Shares for the period. The following table sets forth, for the period indicated, the number of Equity Shares traded on the days high and low prices of our Equity Shares was recorded on NSE for the fiscal years 2004 and 2003. Fiscal Year High Date Number of Low Date No of Shares Traded Shares Traded 2003 28/03/03 3,023,682 22/05/02 173,312 2004 30/03/04 5,221,078 1/04/03 2,373,847 The following table sets forth, for the period indicated, the number of Equity Shares traded on the days high and low prices of our Equity Shares was recorded on NSE for the last six months preceding the date of filing of this Red Herring Prospectus with SEBI. Month High Date Number of Low Date No of Shares Shares Traded Traded August 2004 09/08/04 1,044,560 23/08/04 1,213,332 September 2004 07/09/04 728,156 28/09/04 1,051,120 October 2004 05/10/04 1,011,537 25/10/04 353,771 November 2004 30/11/04 3,439,978 01/11/04 1,081,704 December 2004 31/12/04 1,241,595 01/12/04 3,085,911 January 2005 04/01/05 2,458,219 19/01/05 1,245,715 65 The following table sets forth, for the period indicated, the total volume of Equity Shares traded on the NSE during the fiscal years 2004 and 2003: Fiscal Year Number of Shares Traded 2003 255,255,716 2004 887,976,927 The following table sets forth, for the period indicated, total volume of Equity Shares traded on the NSE during the six months preceding the date of filing with the Designated Stock Exchange. Month Number of Shares Traded August 2004 28,957,308 September 2004 26,262,536 October 2004 12,608,057 November 2004 31,467,039 December 2004 36,003,817 January 2005 37,829,919 Promise vs. Performance We have not made any projections in the offer document of our previous capital issue during the last five years. The funds raised from the capital issue have been utilized for our business as mentioned in the offer documents. Servicing Behaviour There has been no default in payment of statutory dues or of interest or principal in respect of our borrowings or deposits. Other Disclosures The market price of our Equity Shares on September 10, 2004, the day prior to the day our Board of Directors approved the Issue, was Rs. 276.20. On September 11, 2004, the day our Board of Directors approved the Issue, the Stock Exchanges were closed and there was no trading. Our Equity Shares are actively traded on the BSE and the NSE. There are no shares held/sold by the Directors and/or their Relatives and Associates during six months from the date of filing of this Red Herring Prospectus with the Designated Stock Exchange. 66 SELECTED FINANCIAL INFORMATION The following table sets forth selected financial information derived from our unconsolidated financial statements. The financial information as of and for the fiscal years ended March 31, 2000, 2001, 2002, 2003 and 2004 and as of and for the six months ended September 30, 2003 and 2004 is based on our restated financial statements for those periods, which are included in this Red Herring Prospectus under the section titled “Financial Statements” on page F-1. The restated financial statements have been prepared in accordance with Indian GAAP and the SEBI Guidelines and have been restated as described in the auditors’ report attached thereto. The selected financial information presented below should be read in conjunction with the financial statements included in this Red Herring Prospectus, the notes thereto and the sections titled “Management’s Discussion and Analysis of Financial Condition and Results of Operations”on page 90 and “Recent Developments” on page 117. Indian GAAP differs in certain significant respects from U.S. GAAP. For more information on these differences, see the section titled “Summary of Significant Differences between Indian GAAP and U.S. GAAP”, on page 270. SELECTED PROFIT AND LOSS INFORMATION (In Rs. million) Fiscal year First six months of fiscal year 2000 2001 2002 2003 2004 2004 2005 INCOME Interest Income Interest and discount on advances and bills 25,142.3 28,235.4 33,178.8 37,115.9 38,760.1 19,319.9 20,372.4 Income on investments 24,171.0 27,417.8 30,027.6 32,983.3 36,809.7 17,810.9 19,706.4 Interest on balance with RBI and other interbank lending 1,715.4 1,522.9 2,276.3 1,793.8 1,131.3 561.9 656.1 Interest on income tax 168.6 896.8 163.8 2,152.7 191.8 8.4 76.2 Others 348.2 561.9 832.2 804.4 904.1 467.8 260.1 Total interest income 51,545.5 58,634.8 66,478.7 74,850.1 77,797.0 38,168.9 41,431.2 Other Income Commission, exchange and brokerage 3,817.1 4,193.5 4,339.9 4,800.1 5,519.0 2,498.2 3,218.0 Profit on sale of investments (net) 2,149.2 2,420.3 4,379.1 6,722.8 12,363.7 6,989.8 2,786.1 Profit (loss) on revaluation of investments (net) (0.1) (257.5) (581.7) (700.8) (1,178.0) (7.2) (520.8) Profit on sale of land, buildings and other assets (net) 6.8 3.7 3.7 4.1 2.4 1.4 3.8 Profit on exchange transactions (net) 761.6 941.5 922.4 950.8 1,060.2 556.7 669.5 Income from dividends, etc. 202.5 30.0 280.0 237.0 303.0 183.0 163.0 Miscellaneous income 339.5 452.7 433.8 489.1 598.5 268.4 4,183.7 Total other income 7,276.6 7,784.2 9,777.2 12,503.1 18,668.8 10,490.3 10,503.3 Total Income 58,822.1 66,419.0 76,255.9 87,353.2 96,465.8 48,659.2 51,934.5 67 (In Rs. million) Fiscal year First six months of fiscal year 2000 2001 2002 2003 2004 2004 2005 EXPENDITURE Interest Expenditure Interest on deposits 33,667.9 36,094.6 41,216.2 41,625.6 39,264.4 19,744.6 21,006.4 Interest on RBI and interbank borrowings 290.7 392.3 478.9 75.3 133.4 34.7 174.1 Others 1,423.4 1,763.6 1,830.7 1,912.0 2,152.1 1,078.9 1,199.2 Total interest expenditure 35,382.0 38,250.5 43,525.8 43,612.9 41,549.9 20,858.2 22,379.7 Operating Expenses Payment to and provision for employees and wages 11,836.7 14,590.8 13,163.2 14,760.8 16,540.6 7,602.6 9,515.6 Rent, taxes and lighting 719.8 824.1 943.1 1,055.7 1,199.5 580.3 644.9 Printing and stationery 223.4 244.5 285.5 525.1 350.9 169.2 178.9 Advertisement and publicity 43.4 47.1 84.2 78.4 108.5 35.4 58.6 Depreciation on bank properties (net of amounts adjusted against revaluation reserve) 429.9 746.3 852.3 1,289.8 1,814.5 658.8 705.0 Directors’ fees, allowances and expenses 1.2 1.7 3.1 2.8 3.5 1.5 2.6 Auditors’ fees and expenses 72.0 88.5 133.1 155.7 178.8 73.6 100.7 Law charges 58.4 60.3 76.1 85.8 132.5 61.9 52.7 Postage, telegrams, telephones, etc. 236.6 256.7 285.0 313.1 451.1 197.2 256.2 Repairs and maintenance 136.8 169.7 189.8 199.2 244.2 115.6 136.0 Insurance 305.5 331.1 388.8 461.0 546.6 272.7 413.5 Other expenditure 1,174.8 1,355.6 1,587.9 1,639.9 2,136.6 934.7 1,132.8 Total operating expenses 15,238.5 18,716.4 17,992.1 20,567.3 23,707.3 10,703.5 13,197.5 Total Expenditure 50,620.5 56,966.9 61,517.9 64,180.2 65,257.2 31,561.7 35,577.2 Gross profit before provisions and contingencies 8,201.6 9,452.1 14,738.0 23,173.0 31,208.6 17,097.5 16,357.3 Provisions and Contingencies Provision for non performing advances 1,974.6 3,258.0 3,913.3 4,729.7 4,010.4 2,405.2 1,182.1 Provision for standard accounts 515.0 147.0 150.0 140.0 465.0 50.0 130.0 Floating provision against NPAs 0.0 0.0 2,400.0 3,600.0 7,930.0 5,730.0 — Depreciation on investments (77.9) 200.8 (352.1) 1,327.5 (308.6) (177.0) (429.0) 68 (In Rs. million) Fiscal year First six months of fiscal year 2000 2001 2002 2003 2004 2004 2005 Provisions for income tax 1,240.0 1,108.3 1,983.5 3,406.6 6,599.9 2,803.5 2,831.2 Provision for restructured standard accounts 0.0 0.0 155.3 771.1 1,207.4 510.0 789.5 Others 468.5 101.6 864.1 776.1 217.6 260.6 4,501.3 Total provisions and contingencies 4,120.2 4,815.7 9,114.1 14,751.0 20,121.7 11,582.3 9,005.1 Net profit 4,081.4 4,636.4 5,623.9 8,422.0 11,086.9 5,515.2 7,352.2 ADJUSTMENTS ON ACCOUNT OF RESTATEMENT Adjustments on account of changes in accounting policies: Amortization of goodwill on account of amalgamation of Nedungadi Bank — — — (373.3) 373.3 186.7 — Impact of wage revision — — — (460.0) (1,104.0) (552.0) 861.0 Tax impact of adjustments — — — 306.2 254.9 127.5 (303.9) Total adjustments — — — (527.1) (475.8) (237.8) 557.1 Transfer from other reserves ———— ——584.9 Adjusted net profit 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 8,494.2 APPROPRIATIONS Transfer to statutory reserve 1,020.3 1,159.1 1,406.0 2,105.5 2,771.7 — 967.7 Transfer to revenue and other reserve 2,457.5 2,886.4 1,405.7 2,841.5 1,970.1 — 2,903.0 Transfer to investment fluctuation reserve 36.0 0.0 1,755.8 1,900.0 4,648.5 — — Transfer to capital reserve 0.0 6.1 419.7 0.4 23.6 — — Dividends to central government and public 500.0 530.7 636.7 928.5 1,061.2 — — Dividend tax 67.6 54.1 0.0 119.0 136.0 — — Balance carried over to balance sheet 0.0 0.0 0.0 0.0 0.0 5,277.4 4,623.5 Total appropriations 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 8,494.2 69 SELECTED BALANCE SHEET INFORMATION (In Rs. million) As of March 31, As of September 30, 2000 2001 2002 2003 2004 2004 2005 ASSETS Cash in hand 4,281.3 4,452.0 5,777.7 6,288.1 6,992.1 5,659.8 5,570.0 Cheques and demand drafts in hand 0.0 0.0 1,532.5 0.0 0.0 0.0 0.0 Balance with RBI 50,492.7 49,204.2 43,704.1 59,397.1 60,430.7 41,429.6 60,846.4 Balance with banks 7,525.8 7,017.3 11,150.7 14,322.4 13,811.9 9,978.8 14,107.6 - In India 1,313.8 2,813.1 5,992.6 13,547.8 12,816.3 5,899.3 13,804.8 - Outside India 6,212.0 4,204.2 5,158.1 774.6 995.6 4,079.5 302.8 Money at call and short notice 0.0 18.6 1,818.3 764.2 6,970.4 6,759.2 50.6 Investments 220,990.7 251,284.2 282,071.7 340,300.5 421,254.9 419,269.3 460,185.8 Advances 225,717.2 280,290.5 343,694.2 402,281.2 472,247.2 401,968.2 518,705.4 Fixed Assets 6,805.3 7,239.2 7,956.1 8,473.7 8,998.4 8,787.8 9,084.2 Less: Revaluation Reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 Net Fixed Assets 3,253.1 3,772.5 4,574.8 5,177.9 5,788.0 5,534.7 5,916.6 Other Assets 25,472.8 35,545.1 31,441.3 30,323.7 33,172.9 32,554.1 32,300.5 Total Assets (A) 537,733.6 631,584.4 725,765.3 858,855.1 1,020,668.1 923,153.7 1,097,682.9 LIABILITIES Deposits 474,832.3 561,311.3 641,234.8 758,135.0 879,163.9 801,848.9 945,886.4 - Demand deposits from banks 1,518.1 1,477.2 1,112.5 1,660.2 1,431.8 1,438.1 929.9 - Demand deposits from others 52,851.9 61,636.7 66,471.6 97,222.7 97,572.0 81,190.0 96,552.2 - Savings deposits 158,754.1 185,300.6 216,642.1 256,478.9 304,226.1 279,108.6 330,151.0 - Term deposits from banks 9,778.3 12,424.0 13,161.7 4,847.8 7,178.2 4,592.5 3,512.9 - Term deposits from others 251,929.9 300,472.8 343,846.9 397,925.4 468,755.8 435,519.7 514,740.4 Borrowings 6,624.3 6,732.0 4,085.7 6,621.6 12,890.6 7,452.4 19,520.2 - In India 6,561.3 6,661.0 4,041.7 5,608.9 6,260.0 4,153.7 6,405.1 - Outside India 63.0 71.0 44.0 1,012.7 6,630.6 3,298.7 13,115.1 Other liabilities and provisions 28,605.6 31,417.3 38,220.7 41,662.9 59,130.2 53, 505.5 55,033.6 Subordinated debt 8,497.7 8,898.5 11,798.6 15,928.6 23,578.6 18,578.6 23,428.6 Total liabilities (B) 518,559.9 608,359.1 695,339.8 822,348.1 974,763.3 881,385.5 1,043,868.8 NET ASSETS (C=A-B) 19,173.7 23,225.3 30,425.5 36,507.0 45,904.8 41,768.2 53,814.1 70 (In Rs. million) As of March 31, As of September 30, 2000 2001 2002 2003 2004 2004 2005 Share capital 2,122.4 2,122.4 2,122.4 2,653.0 2,653.0 2,653.0 2,653.0 Share application money 0.0 0.0 1,644.9 0.0 0.0 0.0 0.0 Total share capital (D) 2,122.4 2,122.4 3,767.3 2,653.0 2,653.0 2,653.0 2,653.0 RESERVES AND SURPLUS Statutory reserve 4,468.1 5,627.2 7,033.2 9,138.7 11,910.4 9,138.7 12,878.1 Capital reserve 130.5 136.6 556.2 556.6 580.3 556.6 580.3 Revaluation reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 Investment fluctuation reserve 1,345.4 1,345.4 3,101.3 5,001.3 9,649.8 5,001.3 9,649.8 Revenue and other reserve 11,107.3 13,993.7 15,399.3 17,474.9 19,428.8 17,458.7 21,746.9 Deferred tax reserve 0.0 0.0 568.2 568.2 568.2 568.2 568.2 Balance of profit and loss Account 0.0 0.0 0.0 0.0 0.0 5,277.4 4,623.5 Share premium 0.0 0.0 0.0 1,114.3 1,114.3 1,114.3 1,114.3 Total before revaluation reserve 20,603.5 24,569.6 30,039.5 37,149.8 46,462.2 42,368.3 54,328.7 Less revaluation reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 Total reserves and surplus (E) 17,051.3 21,102.9 26,658.2 33,854.0 43,251.8 39,115.2 51,161.1 TOTAL (D+E) 19,173.7 23,225.3 30,425.5 36,507.0 45,904.8 41,768.2 53,814.1 71 SELECTED STATISTICAL INFORMATION The following information should be read together with our financial statements included in this Red Herring Prospectus as well as the section titled “Management’s Discussion and Analysis of Financial Condition and Results of Operations.” The amounts presented in this section are based on our unconsolidated financial statements prepared in accordance with Indian GAAP and internally generated statistical data. These amounts do not give effect to the adjustment to our net profits as a result of the restatement of our unconsolidated financial statements in connection with this Issue. Average Balance Sheet and Net Interest Margin The table below presents the average balances for interest-earning assets and interest-bearing liabilities together with the related interest income and expense amounts, resulting in the presentation of the average yields and cost for each period. The average balance is the monthly average of balances outstanding, as reported to the RBI on Form X. The average yield on average interest-earning assets is the ratio of interest income to average interest-earning assets. The average cost on average interest-bearing liabilities is the ratio of interest expense to average interest-bearing liabilities. The average balances in the table below include gross non-performing assets. We have not recalculated income that is subject to tax benefits on a tax- equivalent basis. (In Rs. billions, except percentages) Fiscal year 2002 2003 2004 Interest Average Interest Average Interest Average Average income/ yield/ Average income/ yield/ Average income/ yield/ balance expense cost balance expense cost balance expense cost Assets: Advances 310.91 33.18 10.67% 369.51 37.12 10.04% 427.03 38.76 9.08% Investments 274.63 30.03 10.93% 317.83 32.98 10.38% 403.40 36.80 9.09% Others (1)(2) 37.56 3.27 8.71% 33.54 4.75 14.16% 26.49 2.24 8.97% Total interest earning assets 623.10 66.48 10.67% 720.88 74.85 10.38% 856.92 77.80 9.08% Fixed assets 7.57 — — 8.29 — — 8.63 — — Other assets (3) 71.83 — — 78.61 — — 92.89 — — Total assets 702.50 66.48 9.46% 807.78 74.85 9.27% 958.44 77.80 8.12% Liabilities: Deposits 594.06 41.22 6.94% 675.75 41.63 6.16% 783.43 39.26 5.01% Demand deposits (4) 64.78 — — 74.59 — — 83.21 — — Savings deposits 195.24 7.04 3.60% 228.19 8.16 3.57% 269.16 8.48 3.15% Term deposits 334.04 34.18 10.23% 372.97 33.47 8.97% 431.07 30.78 7.14% Borrowings 16.99 2.31 13.59% 17.80 1.99 11.16% 27.88 2.29 8.20% Unsecured subordinated bonds (“Tier 2 bonds”) 11.18 1.61 14.40% 14.60 1.73 11.85% 17.76 1.95 10.98% Other borrowings 5.81 0.70 12.05% 3.20 0.25 7.81% 10.12 0.33 3.26% Total interest bearing liabilities 611.06 43.53 7.12% 693.55 43.61 6.29% 811.32 41.55 5.12% Capital and reserves 32.67 — — 42.06 — — 48.62 — — Other Liabilities 58.78 — — 72.17 — — 98.50 — — Total liabilities 702.51 43.53 6.20% 807.78 43.61 5.40% 958.44 41.55 4.34% Net interest income — 22.95 — — 31.24 — — 36.25 — Net interest margin (5) — — 3.68% — — 4.33% — — 4.23% 72 (In Rs. billions, except percentages) First six months of fiscal year 2004 2005 Interest Average Interest Average Average income/ yield/ Average income/ yield/ balance expense cost balance expense cost Assets: Advances 408.82 19.32 9.45% 498.22 20.73 8.32% Investments 375.21 17.81 9.49% 480.02 19.61 8.17% Others (1) 30.27 1.04 6.86% 23.93 1.09 9.12% Total interest earning assets 814.30 38.17 9.37% 1,002.18 41.43 8.27% Fixed assets 8.57 — — 8.90 — — Other assets (3) 85.60 — — 114.31 — — Total assets 908.48 38.17 8.40% 1,125.39 41.43 7.36% Liabilities: Deposits 748.38 19.74 5.28% 908.97 21.01 4.62% Demand deposits (4) 80.17 — — 91.92 — — Savings deposits 257.44 4.08 3.17% 306.26 4.86 3.17% Term deposits 410.76 15.66 7.63% 510.80 16.14 6.32% Borrowings 25.03 1.11 8.90% 42.34 1.37 6.49% Unsecured subordinated bonds 16.32 0.93 11.46% 23.55 1.14 9.71% Other borrowings 8.71 0.18 4.10% 18.79 0.23 2.44% Total interest bearing liabilities 773.41 20.86 5.39% 951.32 22.38 4.70% Capital and reserves 40.32 — — 53.45 — — Other Liabilities 94.75 — — 120.62 — — Total liabilities 908.48 20.86 4.59% 1,125.39 22.38 3.98% Net interest income — 17.31 — — 19.05 — Net interest margin (5) — — 4.25% — — 3.80% (1) Includes contributions made to Rural Infrastructure Development Fund, interest bearing balances with the RBI and money at call and short notice. (2) The increase in yield of “other” assets in fiscal 2003 was primarily due to a refund of disputed income tax payments attributable to prior years that we received in fiscal 2003, together with the interest thereon. (3) Includes cash, balances with banks and non-interest bearing balances with the RBI. (4) Demand deposits do not bear interest. If this item was excluded from the interest bearing liabilities category, our average cost of interest bearing liabilities for years 2002, 2003 and 2004 and first six months of fiscal 2004 and 2005 would have been 7.97%, 7.05%, 5.71%, 6.02% and 5.2%respectively. (5) The net interest margin is ratio of net interest income to total interest earning assets. 73 Analysis of Changes in Interest Income and Interest Expense Volume and Rate Analysis The following table sets forth, for the periods indicated, the allocation of the changes in our interest income and interest expense between average volume and changes in average rates. The changes in net interest income between periods have been reflected as attributed either to volume or rate changes. For purposes of this table, changes which are due to both volume and rate have been allocated solely to changes in rate. (In Rs. billions) First six months of fiscal 2005 vs. First six months of Fiscal 2003 vs. Fiscal 2002 Fiscal 2004 vs. Fiscal 2003 fiscal 2004 Net Change Change Net Change Change Net Change Change change in due to due to change in due to due to change in due to due to interest change in change in interest change in change in interest change in change in income average average income average average income average average or volume rate or volume rate or volume rate expense (1) (2) expense (1) (2) expense (1) (2) Interest income: Advances 3.94 6.25 (2.31) 1.64 5.78 (4.14) 1.41 4.22 (2.81) Investments 2.95 4.72 (1.77) 3.82 8.88 (5.06) 1.80 4.97 (3.17) Others (1)(2) 1.48 (0.35) 1.83 (2.51) (1.00) (1.51) 0.05 (0.22) 0.27 Total interest earning assets 8.37 10.62 (2.25) 2.95 13.66 (10.71) 3.26 8.97 (5.71) Interest expense Deposits 0.41 5.17 (4.76) (2.37) 6.67 (9.04) 1.26 4.59 (3.33) Savings deposits 1.12 1.19 (0.07) 0.32 1.46 (1.14) 0.78 0.77 0.01 Term deposits (0.71) 3.98 (4.69) (2.69) 5.21 (7.90) 0.48 3.82 (3.34) Borrowings (0.33) 0.18 (0.51) 0.30 0.91 (0.61) 0.26 0.62 (0.36) Unsecured Redeemable Bonds 0.12 0.49 (0.37) 0.22 0.37 (0.15) 0.21 0.41 (0.20) Other borrowings (0.45) (0.31) (0.14) 0.08 0.54 (0.46) 0.05 0.21 (0.16) Total interest bearing liabilities 0.08 5.35 (5.27) (2.07) 7.58 (9.65) 1.52 5.21 (3.69) Net interest income 8.29 5.27 3.02 5.02 6.08 (1.06) 1.74 3.76 (2.02) (1) The change due to change in average volume was calculated from the change in average balance over the two periods multiplied by the average rate in the earlier period. (2) The change due to change in average rate is the total change less the change due to change in volume. (3) Includes contributions made to Rural Infrastructure Development Fund, interest bearing balances with the RBI and money at call and short notice. 74 Yields, Spreads and Margins The following table sets forth, for the periods indicated, the yields, spreads and interest margins on our interest-earning assets. (In Rs. billions, except percentages) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 Interest income 66.48 74.85 77.80 38.17 41.43 Average interest-earning assets 623.10 720.88 856.92 814.30 1,002.18 Interest expense 43.53 43.61 41.55 20.86 22.38 Average interest-bearing liabilities(1) 611.06 693.55 811.32 773.41 951.32 Net interest income 22.95 31.24 36.25 17.31 19.05 Average total assets 702.50 807.78 958.44 908.48 1,125.39 Average interest-earning assets as a percentage of average total assets 88.70% 89.24% 89.41% 89.63% 89.05% Average interest-bearing liabilities as a percentage of average total assets 86.98% 85.86% 84.65% 85.13% 84.53% Average interest-earning assets as a percentage of average interest- bearing liabilities 101.97% 103.94% 105.62% 105.29% 105.35 Yield(2) 10.7% 10.0% 9.1% 9.4% 8.3% Cost of funds(3) 7.1% 6.3% 5.1% 5.4% 4.7% Spread(4) 3.6% 3.7% 4.0% 4.0% 3.6% Net interest margin(5) 3.7% 4.3% 4.2% 4.2% 3.8% (1) Includes demand deposits. (2) Yield of interest earning assets. (3) Cost of interest bearing liabilities. Excludes equity and includes the cost of our Tier 2 bonds. (4) Spread is the difference between yield on average interest-earning assets and cost of funds. (5) Net interest margin is the ratio of net interest income to average interest-earning assets. The difference in net interest margin and spread arises due to the difference in the amount of average interest-earning assets and average interest- bearing liabilities. If average interest-earning assets exceed average interest-bearing liabilities, net interest margin is greater than the spread. If average interest-bearing liabilities exceed average interest-earning assets, net interest margin is less than the spread. 75 Returns on Equity and Assets The following table presents selected financial ratios for the periods indicated. (In Rs. billions, except percentages) Fiscal year First six months of fiscal year 2002 2003 2004 2003 2004 Net profit after tax 5.6 8.4 11.1 5.5 7.4 Average total assets 702.5 807.8 958.4 908.5 1,125.4 Average shareholders’ funds 32.7 42.1 48.6 40.3 53.5 Net profit after tax as a percentage of average total assets 0.8% 1.0% 1.2% 1.2% 1.3% Net profit after tax as a percentage of average shareholders’ funds 17.1% 20.0% 22.8% 27.3% 27.5% Average shareholders’ funds as a percentage of average total assets 4.7% 5.2% 5.1% 4.4% 4.7% The following table sets forth, as of September 30, 2004, an analysis of the residual maturity profile of our investments in coupon-bearing securities. The amounts indicate the book value (i.e. the acquisition cost) of the securities and are gross of depreciation. (In Rs. billions, except percentages) As of September 30, 2004 Up to one One to five Five to ten More than year years years ten years (1) Total Amount Amount Amount Amount Amount Yield Government and other approved securities 12.8 67.9 183.7 110.1 374.5 8.6% Other debt securities 5.8 37.5 17.6 10.0 70.9 7.8% Total coupon-bearing securities 18.6 105.4 201.3 120.1 445.4 8.5% Total market value of coupon-bearing securities — — — — 458.4 — (1) Includes perpetual maturity securities. Funding Our funding operations are designed to optimize the cost of funding and effective liquidity management. The primary source of funding is deposits raised from our customers. 76 Total Deposits The average cost (interest expense divided by average of monthly balances) of savings deposits was 3.6% in fiscal 2003, 3.2% in fiscal 2004 and 3.2% in the first six months of fiscal 2005. The average cost of term deposits was 9.0% in fiscal 2003, 7.1% in fiscal 2004 and 6.32% in the first six months of fiscal 2005. Demand deposits do not bear interest, and are therefore carried at zero cost. The following table sets forth, as of the dates indicated, our outstanding deposits and the percentage composition by each category of deposits. (In Rs. million, except for percentages) March 31, 2003 March 31, 2004 September 30, 2004 Balance Balance Balance outstanding % of total outstanding % of total outstanding % of total Demand deposits 98,882.8 13.0% 99,003.9 11.3% 97,482.1 10.3% Savings deposits 256,479.0 33.9% 304,226.1 34.6% 330,151.0 34.9% Term deposits 402,773.2 53.1% 475,934.0 54.1% 518,253.3 54.8% Total deposits 758,135.0 100.0% 879,164.0 100.0% 945,886.4 100.0% The following table sets forth, as of the dates indicated, the regional exposure of our deposits. (In Rs. million, except for percentages) March 31, 2003 March 31, 2004 September 30, 2004 Geographic Distribution Balance Balance Balance outstanding % of total outstanding % of total outstanding % of total Northern 365.8 48.3% 407.9 46.4% 453.7 48.0% Eastern 95.8 12.6% 108.0 12.3% 105.4 11.1% Central 200.7 26.5% 238.6 27.1% 237.4 25.1% Western 57.2 7.5% 80.7 9.2% 99.8 10.6% Southern 38.6 5.1% 44.0 5.0% 49.6 5.2% Total 758.1 100.0% 879.2 100.0% 945.9 100.0% Total Borrowings The following table sets forth, for the periods indicated, our average outstanding borrowings with and without Tier 2 bonds. (In Rs. billions, except for percentages) Fiscal year 2003 Fiscal year 2004 First six months of fiscal year 2005 Average Interest Average Average Interest Average Average Interest Average balance Expense cost balance Expense cost balance Expense cost Borrowings, excluding Tier 2 bonds 3.20 0.25 7.81% 10.12 0.33 3.26% 18.79 0.23 2.44% Tier 2 bonds 14.60 1.73 11.85% 17.76 1.95 10.98% 23.55 1.14 9.71% Total borrowings 17.80 1.98 11.16% 27.88 2.29 8.20% 42.34 1.37 6.47% 77 Asset-Liability Gap The following tables set forth our asset-liability gap position as of March 31, 2004 and September 30, 2004. (In Rs. billions, except for percentages) As of March 31, 2004 (1) Total 1-14 15-28 29 days >3 to >6 to >1 to >3 to Over days days to 3 6 12 3 5 5 months months months years years years Outflows Capital 2.7 ——— ————2.7 Reserves and surplus 47.4 — — — — — — — 47.4 Deposits (2) 879.2 43.0 8.8 34.8 44.3 71.3 451.6 24.6 200.8 Borrowings 12.9 0.2 1.2 3.7 1.9 1.3 4.6 0.0 0.0 Other liabilities and provisions 97.6 19.2 2.0 3.1 8.7 8.3 17.6 6.7 32.0 A. Total outflows 1,039.8 62.4 12.0 41.6 54.9 80.9 473.8 31.3 282.9 B. Cumulative outflows 62.4 74.4 116.0 170.9 251.8 725.6 756.9 1,039.8 Inflows Cash (3) 7.0 7.0 — — — — — — — Balances with RBI (4) 60.4 0.0 4.2 2.6 3.1 4.8 30.6 1.6 13.5 Balances with other Banks 20.8 12.1 0.8 2.0 0.0 1.1 4.5 0.0 0.3 Investments (performing) (5) (6) (7) 421.3 0.3 5.1 6.0 2.1 3.8 34.1 55.1 314.8 Advances (performing) 452.8 43.2 11.1 33.1 53.2 65.5 144.4 49.8 52.5 Net NPAs (8) 4.5 0.0 0.0 0.0 0.0 0.0 0.0 4.5 0.0 Fixed assets 9.0 ——— ————9.0 Other assets (9) 64.0 8.4 3.9 6.1 5.4 15.0 9.6 8.2 7.4 C. Total inflows 1,039.8 71.0 25.1 49.8 63.8 90.2 223.2 119.2 397.5 D. Asset / liability gap (C-A) 8.6 13.1 8.2 8.9 9.3 (250.6) 87.9 114.6 E. % Asset/liability gap (D as % of A) 13.8% 109.2% 19.7% 16.2% 11.4% (52.9)% 280.8% 40.5% F. Cumulative asset/ liability gap (D cumulative) 8.6 21.7 29.9 38.8 48.1 (202.5) (114.6) (0.0) G. % Cumulative asset / liability gap (F as % of B) 13.8% 29.2% 25.8% 22.7% 19.1% (27.9)% (15.1)% 0.0% 78 (In Rs. billions, except for percentages) As on September, 2004 (1) Total 1-14 15-28 29 days >3 to >6 to >1 to >3 to Over days days to 3 6 12 3 5 5 months months months years years years Outflows Capital 2.7 ——— ————2.7 Reserves and surplus 54.8 — — — — — — — 54.8 Deposits (2) 945.9 48.6 19.9 41.4 64.4 55.4 478.2 20.4 217.6 Borrowings 19.5 0.3 2.4 4.7 2.6 0.1 4.7 4.6 0.1 Other liabilities and provisions 91.8 14.6 1.5 2.3 6.2 8.9 17.1 5.9 35.3 A. Total outflows 1,114.7 63.5 23.8 48.4 73.2 64.4 500.0 30.9 310.5 B. Cumulative outflows 63.5 87.3 135.7 208.9 273.3 773.3 804.2 1,114.7 Inflows Cash (3) 5.6 5.6 — — — — — — — Balances with RBI (4) 60.9 0.0 5.0 2.9 4.1 3.5 30.2 1.6 13.6 Balances with other Banks 14.1 8.5 0.0 0.0 1.1 0.0 4.5 0.0 0.0 Investments (performing) (5) (6) (7) 459.8 2.1 1.7 3.7 2.5 17.1 29.1 75.3 328.3 Advances (performing) 501.6 56.8 27.8 38.7 44.2 62.0 144.2 45.8 82.1 Net NPAs (8) 1.9 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 Fixed assets 9.1 0.0 — — — — — — 9.1 Other assets (9) 61.7 8.0 2.4 7.3 5.6 16.9 3.5 8.2 9.8 C. Total inflows 1,114.7 80.9 36.9 52.6 57.5 99.6 211.5 132.8 442.9 D. Asset / liability gap (C-A) 17.4 13.1 4.2 (15.7) 35.2 (288.5) 101.9 132.4 E. % Asset/liability gap (D as % of A) 27.4% 55.0% 8.7% (21.4%) 54.7% (57.7%) 329.8% 42.6% F. Cumulative asset/ liability gap (D cumulative) 17.4 30.5 34.7 19.0 54.2 (234.3) (132.4) 0.0 G. % Cumulative asset / liability gap (F as % of B) 27.4% 34.9% 25.6% 9.1% 19.8% (30.3%) (16.5%) 0.0% (1) Assets and liabilities are classified in the applicable maturity buckets based on residual maturity unless specifically mentioned. 79 (2) Deposit maturities are prescribed by the RBI for demand and savings deposits. For term deposits, we have allocated the maturity by the given term of the account, taking into consideration the projected renewal rate based on previous trends. (3) Cash on hand is classified in the 1-14 day maturity category. (4) Cash and cash equivalents include balances with the RBI to satisfy cash reserve ratio requirements. These balances are held in the form of overnight cash deposits but we classify these balances in the applicable maturity buckets on a basis proportionate to the classification of related deposits. (5) Securities in the trading book are classified in the 1-14 day maturity bucket. (6) Securities held towards satisfying the statutory liquidity ratio (SLR) prescribed by the RBI are classified in the applicable maturity buckets. (7) Shares and units of open ended mutual funds are classified in the 1-14 day maturity category. (8) Net non-performing loans are classified in the >3 to 5 years bucket for substandard assets and over 5 years for all other assets. These net non-performing loans are net of the float provision. (9) Other assets include interest accrued on advances which is placed in the 1-14 day maturity category, interest accrued on investments as per due dates, tax assets allocated on the timetable of refunds expected and others as the inflow is expected. Loan Portfolio As of September 30, 2004, our total outstanding loan portfolio was Rs. 518.7 billion. The following table sets forth, for the dated indicated, our loan portfolio classified by product group. (In Rs.billions, except percentages) March 31, September 30, 2002 2003 2004 2004 Amount % Amount % Amount % Amount % Bills Purchased/Discounted 24.6 7.2 28.8 7.2 28.0 5.9 25.4 4.9 Cash Credit, overdrafts and others 184.8 53.8 209.0 52.0 218.1 46.2 226.9 43.7 Term Loans 134.3 39.0 164.5 40.8 226.1 47.9 266.4 51.4 Total 343.7 100.0 402.3 100.0 472.2 100.0 518.7 100.0 The following table presents our outstanding loans by sector and the proportion of these loans to our outstanding total loans, as of the dates indicated: (In Rs. billions, except percentages) March 31, 2003 March 31, 2004 September 30, 2004 Sector Loans % of total Loans % of total Loans % of total Corporate and Commercial 265.9 66.1% 287.1 60.8% 311.3 60.0% of which Small Scale Industries (SSIs)(1) 47.9 11.9% 57.0 12.1% 57.3 11.0% Retail 65.6 16.3% 91.5 19.4% 99.7 19.2% Agriculture(1) 70.8 17.6% 93.6 19.8% 107.7 20.8% Total outstanding loans 402.3 100.0% 472.2 100.0% 518.7 100.0% (1) As of the last reporting Friday of the month indicated. 80 Concentration of Loans and Credit Substitutes Under the RBI guidelines, our exposure to individual borrowers must not exceed 15.0% of capital funds comprising Tier 1 and Tier 2 capital. Exposure to individual borrowers may exceed the exposure norm of 15.0% of capital funds by an additional 5.0% (i.e., up to 20.0%) provided the additional exposure is on account of infrastructure financing. Under the RBI guidelines, exposure to a group of companies under the same management control must not exceed 40.0% of capital funds unless the exposure is in respect of an infrastructure project in which case the ceiling is 50.0%. Our prudential limits are more conservative and require that not more than 12.5% of capital funds (or 15% for infrastructure) for exposure to an individual borrower, and not more than 30% of capital funds (or 40% for infrastructure) for exposure to a group of companies under the same management. For the purposes of measuring these limits, exposure is calculated based on the total fund based (e.g., loans and advances) and non- fund based (e.g., letters of credit and guarantees) credit as well as investments. Either the credit limit or outstanding amount, whichever is higher, is used when computing the exposure ceiling. The following table sets forth, for the dates indicated, our ten largest single and group exposures as determined by the RBI guidelines, which includes principal outstanding, interest and other dues on advances, investments and 100% of the outstanding amount of non-fund based exposure or the limit, whichever is higher. (In Rs. billions, except percentages) March 31, 2004 September 30, 2004 Exposure % of total % of Exposure % of total of outstanding capital outstanding capital exposure funds exposure funds Borrower 1 21.2 2.1% 32.9% 18.4 1.6% 28.6% Borrower 2 9.2 0.9% 14.3% 13.7 1.2% 21.3% Borrower 3 12.1 1.2% 18.8% 11.5 1.0% 17.8% Borrower 4 10.5 1.0% 16.3% 10.6 0.9% 16.5% Borrower 5 9.8 1.0% 15.2% 9.6 0.9% 15.0% Borrower 6 10.2 1.0% 15.8% 9.2 0.8 % 14.3% Borrower 7 8.5 0.8% 13.1% 9.2 0.8% 14.2% Borrower 8 8.8 0.9% 13.6% 9.2 0.8% 14.2% Borrower 9 9.2 0.9% 14.3% 9.2 0.8% 14.2% Borrower 10 6.4 0.6% 9.9% 8.9 0.8% 13.7% Total 105.9 10.4% 164.2% 109.5 9.6% 169.8% Our internal policies limit our credit exposure to any particular industry to 10.0% of our total credit. The following table sets forth, for the periods indicated, our ten largest fund-based industry exposures based on accounts with total credit limits (fund and non-fund based), or total outstandings (fund and non-fund based) of Rs. 10 million and above. The exposure to the relevant industry is calculated by adding the fund-based outstandings of the accounts in each industry of Rs. 10 million or above. The table sets forth the percentage of each of the top ten industry exposures to our total credit exposure in respect of accounts larger than Rs. 10 million as of the dates indicated. 81 (In Rs. billions, except percentages) March 31, September 30, 2004 2003 2004 Industry Exposure % of Exposure % of Exposure % of exposure exposure exposure Infrastructure(1) 18.4 7.6% 24.3 8.3% 33.1 9.9% Electricity (State Electricity Boards) 14.6 6.0% 21.3 7.2% 31.0 9.3% Iron and steel 22.7 9.4% 23.1 7.9% 27.3 8.2% Financial services 11.3 4.6% 25.6 8.7% 23.3 7.0% Housing 9.0 3.7% 17.9 6.1% 20.6 6.2% Textiles (other than cotton and jute) 14.4 5.9% 17.5 6.0% 18.5 5.6% Engineering 14.9 6.2% 14.9 5.1% 16.7 5.0% Petroleum 9.8 4.1% 4.1 1.4% 16.3 4.9% Food processing 9.2 3.8% 10.4 3.5% 12.5 3.9% Cotton textiles 8.1 3.3% 10.3 3.5% 8.5 2.5% Total 132.4 54.6% 169.4 57.7% 207.8 62.5% (1) Consists of private power projects, telecommunications, roads, ports and others. The following table shows the percentage share of the ten largest borrowers in the following industries of the total exposure of the respective industry as of September 30, 2004. Industry (In Rs. billions, except percentages) Industry Total Industry % exposure of exposure top 10 borrowers to the Industry Infrastructure(1) 33.1 76.2 Electricity (State Electricity Boards) 31.0 87.7 Iron and steel 27.3 42.3 Financial services 23.3 89.6 Housing 20.6 93.7 Textiles (other than cotton and jute) 18.5 24.8 Engineering 16.7 19.0 Petroleum 16.3 100.0 Food processing 12.5 31.2 Cotton textiles 8.5 44.0 Total 207.8 65.4 82 Security The table below shows the amount of our advances which are secured or covered by guarantees, as of the dates indicated. (In Rs. billions, except percentages) March 31, September 30, 2003 2004 2004 Amount % of Amount % of Amount % of advances advances advances Secured by tangible assets (including advances against book debts) 367.1 91.3% 421.2 89.2% 449.3 86.6% Covered by bank or Government guarantees 6.6 1.6% 3.6 0.8% 5.7 1.1% Unsecured 28.6 7.1% 47.4 10.0% 63.7 12.3% Total 402.3 100.0% 472.2 100.0% 518.7 100.0% Non-Performing Assets As of September 30, 2004, our gross non-performing assets as a percentage of gross advances was 7.65% and our net non- performing assets as a percentage of net advances was 0.30%. We define net NPAs as gross NPAs less our loan loss provision, interest in suspense, Deposit Insurance and Credit Guarantee Corporation/Export Credit Guarantee Corporation (DICGC/ECGC) claims received and held and any partial payments received and held. We have made such provisions for 96.4% of our gross non-performing loans. All of our non-performing loans are rupee-denominated. As of September 30, 2004, 45.9% of our gross NPAs were from priority sector advances. We also have significant exposure in the iron and steel and textile industries, which have experienced significant difficulties in recent years, and 31.9% of our non-performing assets are represented by these two industries. Our merger with Nedungadi Bank in February 2003 adversely impacted our non-performing loan portfolio. Nedungadi Bank had gross non-performing assets of Rs. 5.2 billion which was 55.4% of its gross advances as of the date of the merger February 1, 2003. The following table set forth, as of the dates indicated, information about our non-performing loan portfolio: (In Rs. billions, except percentages) March 31, September 30, 2002 2003 2004 2004 NPAs NPAs NPAs NPAs % of % of Gross % of Gross % of Avances NPAs advances Avances NPAs advances Avances NPAs advances Avances NPAs advances Total gross 363.8 41.4 11.39% 429.9 49.8 11.58% 499.5 46.7 9.35% 542.5 41.5 7.65% Derecognized interest and suspended interest 3.5 3.5 — 5.5 5.5 — 4.7 4.7 — 4.0 4.0 — Provisions (as prescribed by RBI) 16.6 16.6 — 22.4 22.4 — 22.6 22.6 — 20.5 20.5 — As reported on balance sheet 343.7 21.3 6.26% 402.3 22.2 5.52% 472.2 19.4 4.11% 518.7 17.0 3.28% Float provision(2) 2.4 2.4 — 6.0 6.0 — 13.9 13.9 — 14.6 14.6 — DICGC/ ECGC held(1) 0.8 0.8 — 0.9 0.9 — 1.0 1.0 — 0.9 0.9 — Net 340.5 18.1 5.32% 395.4 15.3 3.86% 457.3 4.5 0.98% 503.2 1.5 0.30% 83 (1) Represents claims received and held from Deposit Insurance and Credit Guarantee Corporation/Export Credit Guarantee Corporation. (2) The float provision as of September 30, 2004 includes a provision of Rs. 0.70 billion we have built in anticipation of potential changes in the RBI’s NPA norms. Classification of Assets We classify and account for our assets in accordance with the RBI guidelines. Under these guidelines, term loans are regarded as non-performing if any amount of interest or principal remains overdue for more than 90 days; overdrafts and cash credits are regarded as non-performing if the account balance remains out of order for a period of 90 days; and bills are regarded as non- performing if the account remains overdue for more than 90 days. Prior to fiscal 2004, these assets were deemed non- performing if the irregularity continued for 180 days. Prior periods have not been restated to reflect this. In respect of agricultural loans the loan is classified as non-performing if any instalment of principal or interest thereon remains overdue for two crop seasons for short duration crops or one crop season for long duration crops. Crops with crop season longer than one year are long duration crops, and other crops are treated as short duration crops. Our assets are classified as described below: Standard assets Assets that do not disclose any problems or which do not carry more than the normal risk attached to the business of the borrower. Sub-standard assets Assets that are non-performing for a period not exceeding 12 months (or 18 months for fiscal 2004 and prior periods). Doubtful assets Assets that are non-performing for more than 12 months (or 18 months for fiscal 2004 and prior periods). Loss assets Assets (i) with identified losses or (ii) that are considered uncollectible. The following table provides a break down of our gross advances as of the dates indicated. (In Rs. billions, except percentages) March 31, September 30, 2002 2003 2004 2004 Rs. % Rs. % Rs. % Rs. % Standard assets 322.4 88.62% 380.1 88.42% 452.8 90.65% 501.6 92.36% Non-Performing assets 41.4 11.38% 49.8 11.58% 46.7 9.35% 41.5 7.64% of which: Sub-standard 11.1 3.05% 11.2 2.61% 9.1 1.81% 7.2 1.33% Doubtful assets 24.0 6.61% 28.9 6.72% 29.0 5.80% 26.1 4.81% Loss assets 6.3 1.71% 9.7 2.25% 8.7 1.74% 8.2 1.51% Total Loan assets 363.8 100.0% 429.9 100.0% 499.5 100.0% 543.1 100.0% Restructured Assets The RBI has separate guidelines for restructured assets. A fully secured standard asset can be restructured by rescheduling the principal or interest payments, but must be separately disclosed as a restructured asset. The amount of sacrifice, if any, in the element of interest, measured in present value terms, is either written off or provided for to the extent of the sacrifice involved. Similar guidelines apply to sub-standard assets. Sub-standard assets which have been restructured, whether in respect of principal or interest payments, are eligible to be upgraded to the “standard assets” category only after a specified period, which is one year after the date when first payment of interest or of principal, whichever is earlier, falls due, subject to satisfactory performance during the period. 84 In order to create an institutional mechanism for the restructuring of corporate debt, the RBI has also created a corporate debt restructuring (CDR) system in 2003. The objective of this framework is to provide a more timely and transparent mechanism for the restructuring of corporate debts of viable entities facing financial difficulties. This system has led to the approval of restructuring programs for a large number of companies, which has resulted in an increase in the level of restructured assets in the Indian financial system, including an increase in our restructured assets. Following table presents our assets restructured during the periods indicated: (In Rs. billions) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 CDR restructured assets Standard assets — 4.29 7.30 2.54 1.12 Sub-standard assets — 1.35 0.32 — — Doubtful assets — 0.13 1.55 0.20 — Loss assets — — — — 0.24 Total CDR restructured assets — 5.77 9.17 2.74 1.36 Other restructured assets Standard assets 4.83 10.85 3.74 0.61 1.15 Sub-standard assets 1.21 0.17 0.43 0.02 0.05 Doubtful assets 0.00 0.00 0.03 0.01 0.00 Total other restructured assets 6.04 11.02 4.20 0.64 1.20 Total restructured assets 6.04 16.79 13.37 3.38 2.56 Provisioning and Write-offs Our policies on provisioning and write-offs of non-performing assets are consistent with those prescribed by the RBI guidelines. The following is a summary of our provisioning policies: Standard assets A general provision of 0.25%. Sub-standard assets A general provision of 10% and, from the second quarter of fiscal 2005, an additional provision of 10% for advances which were unsecured when made. Doubtful assets We provide for 100% of the unsecured portion of the doubtful assets which are not covered by the realizable value of the security. We provide for secured advances (or the secured portion of partly secured advances) based on the period for which the asset remains doubtful, as follows: Up to one year : 20% provision One to three years : 30% provision More than three years : 50% provision The value assigned to the collateral securing a loan is that reflected on the borrower’s books or that is determined by third party appraisers to be realizable, whichever is lower. Beginning on March 31, 2005, a 100% provision against secured exposure is required for assets categorized as doubtful assets for over three years from April 1, 2004. For assets categorized as doubtful assets for over three years as of March 31, 2004, the provision for 85 secured exposure is to be raised to 60% by March 31, 2005, 75% by March 31, 2006 and 100% by March 31, 2007. Loss assets The entire asset is provided for. Restructured Assets Reductions in the rate of interest, measure in present value terms, is provided for to the extent of the reduction. For the purposes of future interest due as per the original loan agreement in respect of an account, this amount should be discounted to present value at a rate appropriate to the risk category of the borrower and compared to the present value of the amounts expected to be received under the restructuring package discounted on the same basis. The policy regarding asset classification mentioned above is also applicable to restructured assets. In addition to the provisions required by the RBI guidelines, we also make additional float provisions for NPAs. As of March 31, 2004, out of our total provision for NPAs of Rs. 36.5 billion we have Rs. 13.9 billion in excess of the required provision of Rs. 22.6 billion. These provisions account for a coverage ratio for gross NPAs of 90.4% (the coverage ratio is calculated using our total provisions, interest in suspense, Deposit Insurance and Credit Guarantee Corporation/Export Credit Guarantee Corporation (DICGC/ECGC) claims received and held as coverage). This has further increased to 96.4% as on September 30, 2004 by increasing the float provision to Rs. 14.6 billion and providing a build-up provision of Rs. 0.7 billion to meet the revised provisioning norms to be made applicable from March 31, 2005. Provisioning and write -offs have contributed to the reduction in our NPAs. Further, we have sold part of our non-performing assets to an asset reconstruction company, ARCIL against security receipts issued by ARCIL. As of September 30, 2004, we had investments of Rs. 303.7 million in security receipts issued by ARCIL. The price at which the assets were sold was determined on an arms length basis and the sale has resulted in the removal of the non- performing assets from our books. The recovery of principal and the rate of return on the investments in security receipts are dependent on the recovery of the underlying assets. These investments in security receipts are required to be valued at the net asset value as declared by ARCIL. The table below shows the changes in our provisions over the past three years. (In Rs. billions) Fiscal year First six months of fiscal year 2002 2003 2004 2004 2005 Opening Balance 11.9 19.0 28.4 28.4 36.5 ADD: Provisions taken over from Nedungadi Bank amalgamation — 1.4 — — — ADD: Provisions made during the year (including float provisions) 6.8 9.4 14.0 8.7 2.8 LESS: Write-off/write-back during the year. (0.3) 1.4 5.8 1.8 4.2 Closing Balance 19.0 28.4 36.5 35.3 35.1 Once loan accounts are identified as non performing, interest and other fees charged in the account, if uncollected, are suspended. In compliance with regulations governing the presentation of financial information by banks, we report non- performing assets net of cumulative provision and interest suspended in our financial statements. In accordance with the RBI guidelines, interest income from advances for NPAs is recognized upon realization, rather than on an accrual basis as with all other assets. Recovery in respect of non-performing advances is allocated toward interest on the advances, including derecognized or suspended interest, and then towards arrears in principal payments. 86 Sector-wise Analysis of Gross Non-Performing Assets The following table sets forth, as of the dates indicated, the classification of our gross non-performing assets into priority and other sectors. (In Rs. billions, except percentages) March 31, September 30, 2002 2003 2004 2004 Gross % of Gross % of Gross % of Gross % of avances NPAs NPAs avances NPAs NPAs avances NPAs NPAs avances NPAs NPAs Agricultural 50.1 4.4 8.86% 60.8 5.1 8.43% 82.8 4.7 5.72% 95.1 4.2 4.37% SSI 43.2 6.9 15.89% 46.5 9.3 19.98% 56.0 9.3 16.58% 59.3 9.8 16.53% Other priority sector 48.1 6.0 12.41% 64.5 6.0 9.27% 80.8 5.8 7.14% 83.2 5.1 6.16% Total priority sector 141.4 17.3 12.22% 171.8 20.4 11.88% 219.6 19.8 9.01% 237.6 19.1 8.03% Total non-priority sector 222.4 24.1 10.84% 258.1 29.4 11.39% 279.9 26.9 9.61% 305.6 22.5 7.35% Total gross advances 363.8 41.4 11.38% 429.9 49.8 11.58% 499.5 46.7 9.35% 543.2 41.5 7.65% The following table sets forth, as of the dates indicated, the classification of our gross non-performing assets by industry sector. (In Rs. millions, except percentages) March 31, September 30, 2002 2003 2004 2004 % NPAs % NPAs % NPAs % NPAs to adv- to adv- to adv- to adv- Industry NPAs ances Provision NPAs ances Provision NPAs ances Provision NPAs ances Provision Coal 36.3 7.78% 28.5 39.8 31.92% 19.9 21.1 10.30% 17.7 78.5 40.22% 68.4 Mining 530.7 54.73% 376.9 21.1 2.77% 248.3 69.3 4.97% 61.1 120.9 5.97% 104.3 Iron and steel 4,373.0 18.72% 3,768.2 6,789.4 28.31% 3,589.5 7,185.1 30.22% 6,315.3 6,650.4 25.15% 5,782.4 Other metal and metal products 2,227.1 68.84% 1,852.6 1,304.4 28.06% 769.8 957.9 32.37% 834.6 979.7 25.89% 842.1 Engineering 4,114.7 24.75% 3,184.5 3,560.9 23.84% 1,717.3 2,566.3 16.03% 1,979.9 3,265.2 21.68% 2,717.9 Electricity 1,311.0 25.92% 790.3 583.6 9.92% 390.3 437.1 7.78% 157.9 227.1 1.73% 195.5 Cotton textile 2,810.8 31.24% 2,392.4 2,914.6 33.81% 1,141.9 2,615.9 24.56% 2,239.1 2,536.1 24.65% 2,171.5 Jute textile 227.0 14.88% 1,997.5 71.9 9.31% 35.5 79.3 20.96% 30.0 22.8 2.75% 19.5 Other textiles 2,452.5 20.44% 1,526.7 2,165.1 16.84% 1,353.0 2,090.1 16.43% 1,748.7 4,053.1 28.39% 3,503.7 Sugar 283.0 4.46% 135.2 371.0 6.61% 54.8 276.0 3.33% 158.9 165.7 3.13% 144.7 Tea 153.7 8.08% 130.0 35.0 2.00% 65.1 698.1 40.52% 627.3 672.5 26.81% 591.0 Food processing 1,711.9 15.13% 1,285.1 2,237.7 22.49% 1,052.1 1,481.3 14.70% 1,062.8 1,344.1 14.63% 1,140.5 Vegetable oils and vanaspati 847.9 25.61% 393.2 2,238.3 73.76% 1,052.1 732.2 16.14% 394.6 949.7 18.54% 810.9 Tobacco and tobacco products 157.6 59.60% 118.1 32.8 10.73% 14.5 79.7 34.51% 57.1 44.2 18.26% 37.2 Paper and paper products 645.3 15.30% 563.3 676.7 20.17% 308.0 608.3 14.45% 520.1 745.8 21.75% 644.9 Rubber and rubber products 953.2 39.42% 822.8 450.3 24.36% 179.8 387.1 22.51% 336.2 658.7 36.62% 554.6 Chemicals, dyes, paints 369.2 1.97% 1,118.3 3,722.8 18.77% 1,600.5 2,894.0 17.74% 2,275.1 2,281.7 20.47% 1,923.0 Cement 472.9 17.80% 410.2 412.2 13.12% 123.5 300.5 10.52% 257.2 382.3 21.42% 334.3 87 (In Rs. millions, except percentages) March 31, September 30, 2002 2003 2004 2004 % NPAs % NPAs % NPAs % NPAs to adv- to adv- to adv- to adv- Industry NPAs ances Provision NPAs ances Provision NPAs ances Provision NPAs ances Provision Leather and leather products 118.2 3.77% 94.6 295.6 11.29% 119.9 339.1 12.23% 269.8 469.4 17.63% 384.3 Gems and jewelry 235.5 7.71% 124.4 219.9 12.74% 30.0 199.8 7.01% 96.5 113.7 4.51% 97.4 Construction 380.1 9.27% 320.3 831.3 22.25% 411.6 834.9 17.45% 687.1 404.7 1.83% 352.3 Petroleum 1.4 0.03% 1.2 45.9 1.23% 0.0 0.8 0.02% 0.7 60.1 0.39% 51.5 Automobiles including trucks 124.7 4.48% 96.8 657.6 23.14% 403.2 569.2 16.99% 433.0 223.9 11.86% 188.5 Computer software 5.6 0.27% 3.6 461.2 35.39% 204.3 420.8 39.90% 291.0 2.6 0.68% 2.4 Infrastructure 593.7 3.69% 519.6 663.9 5.16% 151.1 654.8 2.97% 585.9 424.9 1.30% 273.5 Other industries 5,657.7 16.50% 3,977.8 4,986.4 14.79% 2,120.8 3,110.8 9.26% 2,320.9 4,613.5 5.96% 3,947.4 Non banking financial companies and trading 1,703.6 5.57% 1,240.7 1,520.8 7.61% 751.9 863.6 3.83% 609.6 796.7 3.73% 562.9 Unclassified advances 8,900.5 6.38% 9,779.9 12,490.5 5.53% 10,444.8 16,228.0 5.84% 12,153.7 9,260.4 3.85% 7,646.2 Total 41,398.6 11.38% 37,052.9 49,800.6 11.58% 28,353.3 46,701.3 9.35% 36,521.7 41,548.4 7.65% 35,092.8 The following table sets forth our ten largest gross non-performing assets as of September 30, 2004. Together, these borrowers constitute 13.41% of our gross NPAs as of September 30, 2004. (In Rs. million) Gross Derecognized DICGC/ Realizable NPA income Provision ECGC Net NPA value of collateral Industry Borrower A 933.5 28.7 817.5 0.0 904.8 1.75 Iron and Steel Borrower B 835.6 48.4 585.7 0.0 787.2 0.0 Iron and Steel Borrower C 835.2 86.8 259.0 0.0 748.4 473.4 Tea and Fertilizer Borrower D 647.0 69.5 579.4 0.0 577.6 0.0 Textiles Borrower E 504.3 0.0 412.8 0.0 504.3 117.04 Electronics Borrower F 395.7 0.0 38.8 0.0 395.7 0.0 Pharmaceuticals Borrower G 401.7 0.0 401.7 0.0 401.7 1,733.7 Iron and Steel Borrower H 368.7 30.6 338.1 0.0 338.1 0.0 Iron and Steel Borrower I 338.9 18.5 188.9 0.0 320.4 0.0 Textiles Borrower J 310.8 27.8 184.9 0.0 283.0 0.0 Pharmaceuticals Total 5,571.4 310.2 3,806.8 0.0 5,261.2 88 Interest in Suspense Interest in suspense is the interest due on non-performing loans that has not been recognized in our books of accounts. The following table sets forth, for the periods indicated, the cumulative amount of interest in suspense on existing non-performing loans. (In Rs. billions) Fiscal year First six months of fiscal year 2001 2002 2003 2004 2004 2005 Interest in suspense 3.2 3.5 5.3 4.7 5.0 4.0 89 MANAGEMENT’S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS You should read the following discussion and analysis of our financial condition and results of operations together with our financial statements included in this report, along with the section titled “Selected Financial Information” beginning on page 67 and the section titled “Selected Statistical Information” beginning on page 72, which presents important statistical information about our business. You should also read the section titled “Risk Factors” beginning on page x, which discusses a number of factors and contingencies that could impact our financial condition and results of operations and the section titled “Recent Developments”on page 117, which discusses our summary financial results for the quarter ended December 31, 2004. The following discussion relates to Punjab National Bank on a standalone basis, and, unless stated otherwise, is based on our restated unconsolidated financial statements which have been prepared in accordance with Indian GAAP and the RBI guidelines. The following discussion is also based on internally prepared statistical information and on information publicly available from the RBI and other sources. A summary of certain differences between our consolidated and unconsolidated results of operations and financial condition is included below. Our fiscal year ends on March 31 of each year so all references to a particular fiscal year are to the twelve months ended March 31 of that year. The six months ended September 30, 2003 and 2004 are referred to herein as the first six months of fiscal 2004 and the first six months of fiscal 2005, respectively. Introduction Overview We are a leading public sector commercial bank in India, offering banking products and services to corporate and commercial, retail and agricultural customers. Our banking operations for corporate and commercial customers include a range of products and services for large corporations, as well as small and middle market businesses and government entities. We offer a wide range of retail credit products including housing loans, personal loans and automobile loans. We cater to the financing needs of the agricultural sector and have created innovative financing products for farmers. We also provide significant financing to other priority sectors including small scale industries. Through our treasury operations, we manage our balance sheet, including the maintenance of required regulatory reserves, and seek to maximize profits from our trading portfolio by taking advantage of market opportunities. Our revenue, which is referred to herein and in our financial statements as our income, consists of interest income and other income. Interest income consists of interest on advances (including the discount on bills discounted) and income on investments. Income on investments consists of interest and dividends from securities and our other investments and interest from interbank loans and cash deposits we keep with the RBI. Our securities portfolio consists primarily of Government of India and state government securities. We meet our statutory liquidity reserve ratio requirements through investments in these and other approved securities. We also hold debentures and bonds issued by public sector undertakings and other corporations, commercial paper, equity shares and mutual fund units. Our interest expense consists of our interest on deposits as well as borrowings. Our interest income and expense are affected by fluctuations in interest rates as well as the volume of activity. Our interest expense is also affected by the extent to which we fund our activities with low-interest or non-interest deposits, and the extent to which we rely on borrowings. Our other income consists principally of commission, exchange and brokerage income, gains and losses on sales and revaluation of investments, and gain or loss on foreign exchange transactions. Commission, exchange and brokerage income includes fee income for credit-related transactional services, such as service charges and processing fees that we charge our accounts and fees for remittance services, documentary credits, letters of credit and letters of guarantee. It also includes commissions on sales of non-funded products such as credit cards and insurance. Our non-interest expense consists principally of operating expenses such as expenses for wages and employee benefits, rent paid on premises, insurance, postage and telecommunications expenses, printing and stationery, depreciation on fixed assets, other administrative and other expenses. Provisioning for non-performing assets, depreciation on investments and income tax is included in provisions and contingencies. We use a variety of indicators to measure our performance. These indicators are presented in tabular form in the section titled “Selected Statistical Information” on page 72. Our net interest income represents our total interest income (on advances and 90 investments) net of total interest expense (on deposits and borrowings). Net interest margin represents the ratio of net interest income to the monthly average of total interest earning assets. Our spread represents the difference between the yield on the monthly average of interest earning assets and the cost of the monthly average of interest bearing liabilities. We calculate average yield on the monthly average of advances and average yield on the monthly average of investments, as well as the average cost of the monthly average of deposits and average cost of the monthly average of borrowings. Our cost of funds is the weighted average of the average cost of the monthly average of interest bearing liabilities. For purposes of these averages and ratios only, the interest cost of the unsecured subordinated bonds that we issue for Tier 2 capital adequacy purposes (“Tier 2 bonds”) is included in our cost of interest bearing liabilities. In our financial statements, these bonds are accounted for as “other liabilities and provisions” and their interest cost is accounted for under other interest expenses. The Indian Economy Our financial condition and results of operations are affected in large measure by general economic conditions prevailing in India. The Indian economy has grown steadily over the past three years. GDP growth was 5.8% in fiscal 2002, 4.0% in fiscal 2003 and 8.5% in fiscal 2004. GDP growth in fiscal 2003 was adversely affected by insufficient rainfall which contributed to a decline in agricultural production. GDP growth picked up in fiscal 2004 due to, among other things, agricultural recovery, resurgence of the industrial sector and continued growth in the services sector. In the first six months of fiscal 2005, GDP growth was 7.0%, compared to 6.9% in the first six months of fiscal 2004. Industrial growth was 3.5% in fiscal 2002, 6.2% in fiscal 2003 and 6.6% in fiscal 2004. In the first six months of fiscal 2005, industrial growth was 7.9%, compared to 6.2% in the first six months of fiscal 2004. The agriculture sector grew by 6.5% in fiscal 2002, declined by 5.2% in fiscal 2003 and grew by 9.6% in fiscal 2004. In the first six months of fiscal 2005, agricultural growth was 1.5%. We have significant exposure to the industrial and agricultural sectors and adverse developments in either or both of these sectors could impact our results. The annual rate of inflation measured by the Wholesale Price Index was 1.6% in fiscal 2002, 6.5% in fiscal 2003 and 4.6% in fiscal 2004. Inflation in the first six months of fiscal 2005 was 6.6% compared to 5.3% in the first six months of fiscal 2004. In its mid-term review of annual policy published on October 26, 2004, the RBI reduced its GDP growth forecast for fiscal 2005 from 6.5-7.0% to 6.0-6.5% as a result of a decline in agricultural growth because of insufficient rainfall and has increased its inflation rate forecast from 5.0 to 6.5%. The average exchange rate of the Indian rupee to one US dollar was Rs. 47.53 in fiscal 2002, Rs. 48.27 in fiscal 2003 and Rs. 45.83 in fiscal 2004. On December 31, 2004, the exchange rate was Rs. 43.58. Foreign exchange reserves were US$131 billion as of December 31, 2004. In the last three fiscal years, there has been a general downward movement in interest rates in India, reflecting global economic conditions and policies of the RBI. The RBI has maintained a policy of assuring adequate liquidity in the banking system and has generally lowered the rate at which it would lend to Indian banks to ensure that borrowers have access to funding at competitive rates. The RBI’s primary motive has been to realign interest rates with the market to facilitate a smooth transition from a government-controlled regime in the early 1990s, when interest rates were heavily regulated, to a more market-oriented interest rate regime. Banks have generally followed the direction of interest rates set by the RBI and have adjusted both their deposit rates and lending rates downwards. 91 The following table sets forth the annual bank rate, repo rate, and the average deposit rates and average prime lending rates of five major public sector banks as of the dates indicated. (as percentages) As of Bank rate Reverse Deposit rate Prime lending Repo rate for 1-3 year rate for 5 term for 5 major public major public sector banks sector banks March 31, 2002 6.50 6.00 7.50-8.50 11.0-12.0 March 31, 2003 6.25 5.00 4.25-6.00 10.75-11.50 March 31, 2004 6.00 4.50 4.00-5.25 10.25-11.00 December 31, 2004 6.00 4.75 5.25-6.25* 10.25-10.75 Relates to rates for major banks for term deposits of more than one year maturity. Source: Reserve Bank of India statistical data. Seasonal trends in the Indian economy affect the banking industry and therefore our business. The period from October to March is the busy period in India for economic activity and accordingly we generally experience high volumes of business during this period. Economic activity in the period from April to September is lower than in the busy period; accordingly, our business volumes are generally lower during this period. Critical Accounting Policies Interest on advances is recognized on an accrual basis except in respect of advances classified as non-performing, where interest income is recognized upon realization. Prior to March 31, 2004, advances were classified as non-performing if any amount of interest or principal remained overdue for more than 180 days. From March 31, 2004, this period was shortened to 90 days. See also the discussion under the section titled “Selected Statistical Information” on page 72. Commissions, interest on overdue bills, income from foreign exchange transactions and merchant banking transactions, income from mutual fund units and other fees are recognized upon realization. In accordance with the RBI guidelines, we classify our investments into three categories. Securities which we intend to hold until maturity are classified as “Held to Maturity” securities. These securities are recorded on our balance sheet at their acquisition cost and any premium paid to acquire these securities is amortized in our statement of profit and loss over the remaining years to maturity of the securities. For the fiscal years discussed herein, these investments were not allowed to exceed 25% of our total investments. Following a change in the RBI guidelines in September 2004, these investments are not allowed to exceed 25% of our net demand and time liabilities. Securities that are held with the intention to trade by taking advantage of short-term price or interest rate movements are classified as “Held for Trading”, and securities not falling into either of the first two categories are classified as “Available for Sale”. Our investments are accounted for under various sub- categories, such as government securities, equity shares, preference shares, debentures and bonds, mutual funds and commercial paper. For “Held for Trading” and “Available for Sale” securities, any appreciation or depreciation in value is aggregated within each sub-category. We provide for any net depreciation in value and ignore any net appreciation in value. Gains or losses on the sale of investments are recognized in our profit and loss account. In addition, the amount of gain on the sale of “Held to Maturity” investments is appropriated to our capital reserve account. On September 24, 2004, we transferred a certain amount of government securities from the “Available for Sale” category to the “Held to Maturity” category. On the date of transfer, in accordance with the RBI guidelines, we recognized a one-time loss on the sale of investments of Rs. 2.1 billion on account of this transfer, representing the consequential depreciation effect of the transfer as on that date. Income from swap transactions that we enter into for hedging purposes are accounted for on an accrual basis. Gains or losses on termination of such swaps are recognized over the shorter of the remaining life of the swap contract or of the hedged asset or liability. Swaps that do not hedge assets or liabilities are carried at the lower of cost or market value and are marked to market on a monthly basis. 92 Our policies on provisioning and write-offs of non-performing assets are consistent with those prescribed by the RBI guidelines. The size of our “floating” provision against non-performing assets, however, is determined by our management based on a number of factors, including our net profit position, tax benefits available, management’s perception of market risk, interest rate outlook, expectations and estimates regarding our asset portfolio and its future performance and general prudential principles. As of March 31, 2004, we had a coverage ratio of 90.4% for our non-performing assets and floating provisions against non- performing assets of Rs. 13.93 billion and as of September 30, 2004, we had a coverage ratio of 96.4% for our non-performing assets and floating provisions against non-performing assets of Rs. 13.93 billion. Our provisioning policies are discussed in further detail in the section titled “Selected Statistical Information” on page 72. For details of the accounting treatment and related impact of the Capital Reduction, see the section titled “Capital Structure” on page 22. Results of Operations First Six Months of Fiscal 2005 Compared to the First Six Months of Fiscal 2004 Our total income increased by 6.7% from Rs. 48.66 billion in the first six months of fiscal 2004 to Rs. 51.93 billion in the first six months of fiscal 2005 and our total expenditure increased by 12.7% from Rs. 31.56 billion in the first six months of fiscal 2004 to Rs. 35.58 billion in the first six months of fiscal 2005. Our gross profit decreased by 4.3% from Rs. 17.10 billion in the first six months of fiscal 2004 to Rs. 16.36 billion in the first six months of fiscal 2005. Our unadjusted net profit increased by 33.2% from Rs. 5.52 billion in the first six months of fiscal 2004 to Rs. 7.35 billion in the first six months of fiscal 2005. Net Interest Income Our net interest income increased by 10.1% from Rs. 17.31 billion in the first six months of fiscal 2004 to Rs. 19.05 billion in the first six months of fiscal 2005. The following table sets forth the components of our net interest income: (In Rs. million) First Six Months of Fiscal 2004 2005 Interest income 38,168.9 41,431.2 Interest expense 20,858.2 22,379.7 Net interest income 17,310.7 19,051.5 The increase in net interest income was a result of an 8.5% increase in total interest income from Rs. 38.17 billion in the first six months of fiscal 2004 to Rs. 41.43 billion in the first six months of fiscal 2005, which offset a 7.3% increase in total interest expense from Rs. 20.86 billion in the first six months of fiscal 2004 to Rs. 22.38 billion in the first six months of fiscal 2005. The increase in total interest income reflected gains in income from investments, as well as the growth of our loan portfolio. The increase in total interest expense reflected primarily the increase in our deposits. Our average interest earning assets increased by 23.1% in the first six months of fiscal 2005 compared to the first six months of fiscal 2004. Our net interest margin declined from 4.2% in the first six months of fiscal 2004 to 3.8% in the first six months of fiscal 2005 and our spread decreased from 4.0% in the first six months of fiscal 2004 to 3.6% in the first six months of fiscal 2005. 93 Interest Income The following table sets forth the components of our interest income: (In Rs. million) First Six Months of Fiscal 2004 2005 Interest income from advances and bills 19,319.9 20,732.4 Income from investments 17,810.9 19,706.4 Interest on balances with the RBI and other interbank lending 561.9 656.1 Other interest income 476.2 336.3 Total interest income 38,168.9 41,431.2 Interest income from advances and bills increased by 7.3% from Rs. 19.32 billion in the first six months of fiscal 2004 to Rs. 20.73 billion in the first six months of fiscal 2005, reflecting an increase in the average volume of advances as a result of the general growth in our business, offset in part by a decline in average yield. Our average volume of advances increased by 21.9% from Rs. 408.82 billion in the first six months of fiscal 2004 compared to Rs. 498.22 billion in the first six months of fiscal 2005. Our average volume of advances to corporate and commercial customers increased by 13.8% from Rs. 278.30 billion in the first six months of fiscal 2004 compared to Rs. 316.75 billion in the first six months of fiscal 2005. This included small scale industry advances, which increased by 18.0% from Rs. 47.85 billion in the first six months of fiscal 2004 compared to Rs. 56.45 billion in the first six months of fiscal 2005 because of our increased focus on expansion in this sector. Our average retail advances increased by 37.7% from Rs. 68.84 billion in the first six months of fiscal 2004 compared to Rs. 94.78 billion in the first six months of fiscal 2005 primarily because lower interest rates made retail loans more attractive to customers, particularly in the housing and consumer goods segment, in line with trends experienced by the banking industry as a whole, combined with increased marketing efforts on our part. Our average volume of agriculture advances increased by 40.5% from Rs. 61.68 billion in the first six months of fiscal 2004 to Rs. 86.69 billion in the first six months of fiscal 2005 due to our increased focus on this sector. Yields on our advances decreased from an average of 9.45% in the first six months of fiscal 2004 to 8.32% in the first six months of fiscal 2005. This decline was due to the general decline in interest rates and increased competition in the banking industry. Investment income increased by 10.6% from Rs. 17.81 billion in the first six months of fiscal 2004 to Rs. 19.71 billion in the first six months of fiscal 2005. Our average volume of investments increased by 27.9% from Rs. 375.21 billion in the first six months of fiscal 2004 to Rs. 480.02 billion in the first six months of fiscal 2005 because the increase in our deposits provided us with an increased amount of funds to invest. The main component of our investment income is interest on government and other approved securities. Income from these securities increased by 13.2% from Rs. 14.42 billion in the first six months of fiscal 2004 to Rs. 16.32 billion in the first six months of fiscal 2005. The increase was primarily a result of a 30.4% increase in the average volume of these investments from Rs. 306.66 billion in the first six months of fiscal 2004 to Rs. 399.85 billion in the first six months of fiscal 2005 offset in part by a decline in yields. Yields from these investments declined from 9.5% in the first six months of fiscal 2004 to 8.6% in the first six months of fiscal 2005, reflecting the general decline in interest rates during this period and the fact that a larger portion of our portfolio in the first half of fiscal 2005 was held in short-term securities such as treasury bills. Investment income from investments other than government and other approved securities stayed almost even at Rs. 3.38 billion in the first six months of fiscal 2004 compared to Rs. 3.39 billion in the first six months of fiscal 2005 despite an increase in the volume of these investments due to the decline in yields which offset the increase in volume. Yields from investments other than government securities declined from 9.7% in the first six months of fiscal 2004 to 7.8% in the first six months of fiscal 2005, reflecting the general decline in interest rates during this period and the fact that a larger portion of our portfolio in 94 the first half of fiscal 2005 was held in short-term securities such as commercial paper. Our investment income in future periods may be adversely impacted as the high coupon securities in our investment portfolio mature. As of September 30, 2004, the weighted average yield on all our fixed coupon securities was 8.5% and their weighted average maturity was 5.3 years. As of that date, central government securities constituted 72.9% of our government securities portfolio and the weighted average yield on our central government and state government securities was 8.1% and 9.1%, respectively, and their weighted average maturity was 6.0 years and 5.1 years, respectively. Interest on balances with the RBI and other interbank lending increased by 17.9% from Rs. 0.56 billion in the first six months of fiscal 2004 to Rs. 0.66 billion in the first six months of fiscal 2005, mainly because of increased interbank lending and because the increase in our deposits required us to maintain higher CRR balances with the RBI. Interest Expense Our interest expense increased by 7.3% from Rs. 20.86 billion in the first six months of fiscal 2004 to Rs. 22.38 billion in the first six months of fiscal 2005, reflecting primarily the increase in our deposits. Our average cost of funds was 5.4% in the first six months of fiscal 2004 and 4.7% in the first six months of fiscal 2005. Our interest expense on deposits increased by 6.4% from Rs. 19.74 billion in the first six months of fiscal 2004 to Rs. 21.01 billion in the first six months of fiscal 2005 as a result of the increase in the volume of our deposits. Our average total deposits increased by 21.5% from Rs. 748.38 billion in the first six months of fiscal 2004 to Rs. 908.97 billion in the first six months of fiscal 2005. Our average cost of deposits declined from 5.3% in the first six months of fiscal 2004 to 4.6% in the first six months of fiscal 2005, reflecting primarily the decline in interest rates. Our other interest expense, which consists mainly of Tier 2 bonds issued by us, increased by 11.1% from Rs. 1.08 billion in the first six months of fiscal 2004 to Rs. 1.20 billion in the first six months of fiscal 2005 as a result of the issuance in March 2004 of Rs. 5.0 billion in new Tier-2 bonds. Our average cost of borrowings (including our Tier 2 bonds) declined from 8.9% in the first six months of fiscal 2004 to 6.5% in the first six months of fiscal 2005 due to declining interest rates. Other Income Our other income remained substantially unchanged at Rs. 10.50 billion in the first six months of fiscal 2005 from Rs. 10.49 billion in the first six months of fiscal 2004. The following table sets forth the components of our other income: (In Rs. million) First Six Months of Fiscal 2004 2005 Commission, exchange and brokerage 2,498.2 3,218.0 Profit on sale of investments (net) 6,989.8 2,786.2 Profit (loss) on revaluation of investments (net) (7.2) (520.8) Profit on sale of land, buildings and other assets (net)‘ 1.4 3.8 Profit on exchange transactions (net) 556.7 669.5 Dividends from subsidiaries, companies and joint ventures 183.0 163.0 Miscellaneous income 268.4 4,183.6 Total other income 10,490.3 10,503.3 Income from commissions, exchange and brokerage increased by 28.8% from Rs. 2.50 billion in the first six months of fiscal 2004 to Rs. 3.22 billion in the first six months of fiscal 2005. The following table sets forth the components of income from 95 commission, exchange and brokerage: (In Rs. million) First Six Months of Fiscal 2004 2005 Remittances 522.8 536.7 Documentary credits and guarantees 525.2 632.5 Bills 450.1 487.4 Commissions on handling Government business such as tax collection and pension disbursements 214.5230.1 Rent on safe deposit vaults 174.6 176.9 Service charges and processing fees 408.5 615.3 Incidental charges on deposit accounts 185.3 481.2 Others 17.2 57.9 Total 2,498.2 3,218.0 Income from commissions, exchange and brokerage increased mainly because of an increase in service charges and processing fees from Rs. 0.41 billion in the first six months of fiscal 2004 to Rs. 0.62 billion in the first six months of fiscal 2005 as a result of increased services to corporate and retail customers, and an increase in incidental charges on deposit accounts (which consist mainly of charges for not maintaining minimum balances) from Rs. 0.19 billion in the first six months of fiscal 2004 to Rs. 0.48 billion in the first six months of fiscal 2005. Net profit on the sale of investments decreased by 60.1% from Rs. 6.99 billion in the first six months of fiscal 2004 to Rs. 2.79 billion in the first six months of fiscal 2005. This decrease was due in part to a 26.0% decrease in the profit on sale of investments from Rs. 7.51 billion in the first six months of fiscal 2004 to Rs. 5.56 billion in the first six months of fiscal 2005. The higher profit in the first six months of fiscal 2004 was partly due to a gain of Rs. 2.93 billion from the repurchase by the Government of some of its outstanding high-coupon securities, which was then used by us to increase our provisioning for non-performing assets. The rest of the decrease in net profit on the sale of investments was primarily due to increased losses from the sale of investments from Rs. 0.52 billion in the first six months of fiscal 2004 to Rs. 2.77 billion in the first six months of fiscal 2005. The higher loss in the first six months of fiscal 2005 was partly due to the transfer of a certain amount of government securities from the “Available for Sale” category to the “Held to Maturity” category, which resulted in a depreciation of Rs. 2.1 billion. Our net profits on the sale of investments may be adversely affected in future periods by interest rate fluctuations, particularly in an environment of rising interest rates. Revaluation of investments resulted in a net loss of Rs. 520.8 million in the first six months of fiscal 2005 compared to a net loss of Rs. 7.2 million in the first six months of fiscal 2004. The increased loss was primarily because until the end of fiscal 2004 we revalued our investments only at the end of the fiscal year and therefore the net loss on revaluation of investments was reflected only in our full year accounts; however, since the beginning of fiscal 2005 we revalue our investments on a daily basis as a result of which they have been reflected in our six month accounts. Net profit from foreign exchange transactions increased by 19.6% from Rs. 0.56 billion in the first six months of fiscal 2004 to Rs. 0.67 billion in the first six months of fiscal 2005, primarily due to a higher volume of foreign exchange trading activities. Dividend income from our subsidiaries and joint ventures decreased by 11.1% from Rs. 0.18 billion in the first six months of fiscal 2004 to Rs. 0.16 billion in the first six months of fiscal 2005, primarily because of higher dividend income in the first six months of fiscal 2004 as the result of an interim dividend from PNB Gilts Ltd. Miscellaneous income increased significantly from Rs. 0.27 billion in the first six months of fiscal 2004 to Rs. 4.2 billion in the first six months of fiscal 2005. The increase was in large part due to a one-time recognition in our profit and loss account of Rs. 96 3.87 billion representing accumulated credits related to unclaimed funds arising from inter-branch transactions that are more than five years old. We received specific permission from the RBI to book this amount. Of this amount, Rs 2.90 billion was appropriated to our revenue and other reserves and Rs. 0.97 billion was appropriated to our statutory reserves. We have set aside a reserve of Rs. 0.24 billion in case future claims are made in respect of the unclaimed funds. Operating Expenses Total operating expenses increased by 23.4% from Rs. 10.70 billion in the first six months of fiscal 2004 to Rs. 13.20 billion in the first six months of fiscal 2005. As a percentage of our total income, operating expenses increased to 25.4% in the first six months of fiscal 2005 compared to 22.0% in the first six months of fiscal 2004. The primary component of our operating expenses was wages and other payments to employees which increased by 25.3% from Rs. 7.60 billion in the first six months of fiscal 2004 to Rs. 9.52 billion in the first six months of fiscal 2005. As a percentage of total income, wages and other payments to employees increased to 18.3% in the first six months of fiscal 2005 from 15.6% in the first six months of fiscal 2004. Our headcount was 58,747 employees as of September 30, 2004 compared to 58,800 employees as of September 30, 2003. Wages and other payments to employees increased mainly because of a Rs. 1.23 billion additional expense for retirement benefits. As of September 30, 2004, this amount is also reflected in our provisions because the funds had not yet been transferred to the benefit trust. We also made a Rs 2.45 billion contingency provision intended for retirement benefits as discussed below, of which we used Rs 1.23 billion in the third quarter of fiscal 2005 and intend to use Rs. 1.22 billion in the fourth quarter of fiscal 2005. Our expenses for rent, taxes and lighting for our premises increased by 10.3% from Rs. 0.58 billion in the first six months of fiscal 2004 to Rs. 0.64 billion in the first six months of fiscal 2005. As a percentage of total income, these expenses were 1.2% in the first six months of fiscal 2004 and the first six months of fiscal 2005. Depreciation expenses on our properties increased by 7.6% from Rs. 0.66 billion in the first six months of fiscal 2004 to Rs. 0.71 billion in the first six months of fiscal 2005. As a percentage of total revenues, these expenses were 1.4% in the first six months of fiscal 2004 and the first six months of fiscal 2005. Gross Profit For the reasons stated above, our gross profit before provisions and contingencies decreased by 4.3% from Rs. 17.10 billion in the first six months of fiscal 2004 to Rs. 16.36 billion in the first six months of fiscal 2005. As a percentage of total income, our gross profit decreased from 35.1% in the first six months of fiscal 2004 to 31.5% in the first six months of fiscal 2005. Provisions and Contingencies The following table sets forth, for the periods indicated, the components of our provisions and contingencies: (In Rs. million) First Six Months of Fiscal 2004 2005 Provision for non-performing assets 2,405.2 1,182.1 Floating provision against non-performing assets 5,730.0 - Provision for standard accounts 50.0 130.0 Depreciation on investments (177.0) (429.0) Provision for income and wealth tax 2,803.5 2,831.2 Others 770.6 5,290.8 Total provisions and contingencies made during the six month period 11,582.3 9,005.1 * Net of recovery in written-off accounts of Rs. 126.9 million in the first six months of fiscal 2004 and Rs. 156.2 million in the first six months of fiscal 2005. 97 Provisions and contingencies made in the first six months of fiscal 2005 decreased by 22.2% to Rs. 9.01 billion compared to Rs. 11.58 billion in the first six months of fiscal 2004. Our provisioning in respect of non-performing assets (which is the sum of the provision for non-performing assets and the floating provision against non-performing assets in the table above) decreased from Rs. 8.14 billion in the first six months of fiscal 2004 to Rs. 1.18 billion in the first six months of fiscal 2005. This was mainly because we made float provisions of Rs. 5.73 billion in the first six months of fiscal 2004 and did not make any float provisions in the first six months of fiscal 2005 and because our non-performing assets decreased owing to higher recoveries. Our recoveries increased significantly in the agriculture sector and were also assisted by the Securitization Act. Our other provisions increased significantly in the first six months of fiscal 2005 to Rs. 5.29 billion from Rs. 0.77 billion in the preceding six month period. This was largely due to provisions of Rs. 2.45 billion for pension and gratuity benefits, Rs. 1.59 billion on account of the upcoming banking industry wage settlement described below and Rs. 0.79 billion for standard restructured accounts. There has been no wage settlement in the banking industry since 1997, although one was due in 2002. Because a wage settlement has been expected to be reached soon, we provided for a 12% wage increase in our financial statements for the period after October 31, 2002, which is when the previous wage settlement expired. Accordingly, we initially made a provision of Rs. 1.59 billion for the first six months of fiscal 2005 and intended to make further provisions of a similar amount over the remaining quarters of fiscal 2005. However, the Indian Banking Association recently reached a broad understanding with banking industry employees that the maximum wage increase will be 13.25%. Because this understanding was reached, we decided to revise our estimated provision for the first six months of fiscal 2005 to Rs. 2.29 billion from Rs. 1.59 billion and the revision is reflected in our restated financial statements for the period. In addition, in our restated financial statements we have allocated the total 2.29 billion provision among the relevant periods, such that Rs. 0.46 billion has been allocated to fiscal 2003, Rs. 1.10 billion to fiscal 2004 and Rs. 0.73 billion to the first six months of fiscal 2005, as described further below under Adjusted Net Profit. As described in the section titled “Recent Developments”on page 117, we made a provision of Rs.1.07 billion on account of the wage settlement in the third quarter of fiscal 2005. Assuming a wage settlement is not reached, we currently expect to make provisions on account of the wage settlement of Rs. 0.36 billion in the fourth quarter of fiscal 2005. The understanding reached with the banking industry employees is not final and binding, and the amount of our provisions is an estimate based on our past experience. The actual expenditure we will have to incur following the wage settlement could vary materially from the amounts we have provided for. Income Tax Our provision for income tax increased by 1.1% from Rs. 2.80 billion in the first six months of fiscal 2004 to Rs. 2.83 billion in the first six months of fiscal 2005. Net Profit As a result of the foregoing factors, our net profit increased by 33.2% from Rs. 5.52 billion in the first six months of fiscal 2004 to Rs. 7.35 billion in the first six months of fiscal 2005. As a percentage of total income, our net profit increased from 11.3% in the first six months of fiscal 2004 to 14.2% in the first six months of fiscal 2005. Our earnings per equity share increased by 33.3% from Rs. 20.79 per equity share in the first six months of fiscal 2004 to Rs. 27.71 per equity share in the first six months of fiscal 2005. Adjusted Net Profit Our adjusted net profit in the first six months of fiscal 2005 was Rs. 8.49 billion, which is an increase of Rs. 1.14 billion from our previously stated net profit of 7.35 billion. This was due in part to the reallocation of the provision in relation to the upcoming industry wage settlement as described above. The after-tax effect of the reduction in the provision applicable to the first six months of 2005 increased our net profit by Rs. 0.56 billion. The other Rs. 0.58 billion of the increase represented the amount that the RBI allowed us to transfer from our reserves to net profit for building additional provisions towards pension and gratuity benefits. Our adjusted net profit in the first six months of fiscal 2004 was Rs. 5.28 billion, which is Rs. 0.24 billion lower than our previously stated net profit of Rs. 5.52 billion. This was due to the allocation to the first six months of fiscal 2004 of 50% of the adjustments made to the net profit for the full fiscal 2004. These adjustments are described below. 98 Fiscal Year Ended March 31, 2004 Compared to the Fiscal Year Ended March 31, 2003 Our total income increased by 10.4% from Rs. 87.35 billion in fiscal 2003 to Rs. 96.47 billion in fiscal 2004 and our total expenditure increased by 1.7% from Rs. 64.18 billion in fiscal 2003 to Rs. 65.26 billion in fiscal 2004. Our gross profit increased by 34.7% from Rs. 23.17 billion in fiscal 2003 to Rs. 31.21 billion in fiscal 2004. Our unadjusted net profit increased by 31.7% from Rs. 8.42 billion in fiscal 2003 to Rs. 11.09 billion in fiscal 2004. Net Interest Income Our net interest income increased by 16% from Rs. 31.24 billion in fiscal 2003 to Rs. 36.25 billion in fiscal 2004. The following table sets forth the components of our net interest income: (In Rs. million) Year ended March 31, 2003 2004 Interest income 74,850.1 77,797.0 Interest expense 43,612.9 41,549.9 Net interest income 31,237.2 36,247.1 The increase in net interest income was a result of a 3.9% increase in total interest income from Rs. 74.85 billion in fiscal 2003 to Rs. 77.80 billion in fiscal 2004, and a 4.7% decrease in total interest expense from Rs. 43.61 billion in fiscal 2003 to Rs. 41.55 billion in fiscal 2004. This reflected significant gains in income from investments, as well as the growth of our loan portfolio and a decline in interest expense on deposits and borrowings. Our average interest earning assets increased by 18.9% in fiscal 2004 compared to fiscal 2003. Our net interest margin declined slightly from 4.3% in fiscal 2003 to 4.2% in fiscal 2004 and our spread decreased from 4.1% in fiscal 2003 to 4.0% in fiscal 2004. Interest Income The following table sets forth the components of our interest income: (In Rs. million) Year ended March 31, 2003 2004 Interest income from advances and bills 37,115.9 38,760.1 Income from investments 32,983.3 36,809.7 Interest on balances with the RBI and other interbank lending 1,793.8 1,131.3 Interest on income tax refund 2,152.7 191.8 Other interest income 804.4 904.1 Total interest income 74,850.1 77,797.0 Interest income from advances and bills increased by 4.4% from Rs. 37.12 billion in fiscal 2003 to Rs. 38.76 billion in fiscal 2004, reflecting an increase in the average volume of advances as a result of the general growth in our business, offset in part by a decline in average yield. Our average volume of advances increased by 15.6% from Rs. 369.51 billion in fiscal 2003 to Rs. 427.03 billion in fiscal 2004. Our average volume of advances to corporate and commercial customers increased by 7.9% from Rs. 264.14 billion in fiscal 2003 to Rs. 284.98 billion in fiscal 2004. This included small scale industry advances, which increased by 17.6% from Rs. 42.46 billion in fiscal 2003 to Rs. 49.95 billion in fiscal 2004 because of our increased focus on expansion in this sector. The overall increase in advances to corporate and commercial customers was limited because of lower borrowing by corporate customers 99 during the fiscal year, in part due to the cheaper access to funds in international markets and the greater use of internal funds by these customers. Our average retail advances increased by 41.1% from Rs. 53.14 billion in fiscal 2003 to Rs. 74.98 billion in fiscal 2004 primarily because lower interest rates made retail loans more attractive to customers, particularly in the housing and consumer goods segment, in line with trends experienced by the banking industry as a whole, combined with increased marketing efforts on our part. Our average volume of agriculture advances increased by 28.4% from Rs. 52.23 billion in fiscal 2003 to Rs. 67.07 billion in fiscal 2004 due to our increased focus on this sector. Yields on our advances decreased from an average of 10.0% in fiscal 2003 to 9.1% in fiscal 2004. This decline was due to the general decline in interest rates and increased competition in the banking industry. Investment income increased by 11.6% from Rs. 32.98 billion in fiscal 2003 to Rs. 36.81 billion in fiscal 2004. Our average volume of investments increased by 26.7% from Rs. 317.80 billion in fiscal 2003 to Rs. 403.40 billion in fiscal 2004 because the increase in our deposits provided us with an increased amount of funds to invest. Income from government and other approved securities increased by 16.2% from Rs. 26.9 billion in fiscal 2003 to Rs. 31.2 billion in fiscal 2004. The increase was primarily a result of a 34.2% increase in the average volume of our these investments from Rs. 248.35 billion in fiscal 2003 to Rs. 333.30 billion in fiscal 2004 offset in part by a decline in yields. Yields from these investments declined from 10.8% in fiscal 2003 to 9.4% in fiscal 2004, reflecting the general decline in interest rates during this period. Investment income from investments other than government and approved securities decreased by 8.4% from Rs. 6.1 billion in fiscal 2003 to Rs. 5.6 billion in fiscal 2004 despite an increase in the volume of these investments because the decline in yields outweighed the increase in volume. Yields from investments other than government securities declined from 8.8% in fiscal 2003 to 8.0% in fiscal 2004, reflecting the general decline in interest rates during this period. The growth in our investment income may be adversely impacted as the high coupon securities in our investment portfolio mature. As of March 31, 2004, the weighted average yield on all our fixed coupon securities was 9.0% and their weighted average maturity was 5.4 years. As of that date, central government securities constituted 73% of our government securities portfolio and the weighted average yield on our central government and state government securities was 8.5% and 9.7%, respectively, and their weighted average maturity was 6.2 years and 5.1 years, respectively. Interest on balances with the RBI and other interbank lending decreased from Rs. 1.79 billion in fiscal 2003 to Rs. 1.13 billion in fiscal 2004 because the RBI reduced the cash reserve ratio (which is a percentage of net demand and time liabilities that banks are required to maintain in current accounts with the RBI), as a result of which we were required to maintain lower deposits with the RBI during the year. Interest on income tax decreased significantly from Rs. 2.15 billion in fiscal 2003 to Rs. 0.19 billion in fiscal 2004 because in fiscal 2003 we received a substantial refund of disputed income tax payments attributable to prior years, together with the interest thereon. Interest Expense Our interest expense decreased by 4.7% from Rs. 43.61 billion in fiscal 2003 to Rs. 41.55 billion in fiscal 2004, reflecting primarily the decrease in interest rates. Our average cost of funds was 6.3% in fiscal 2003 and 5.1% in fiscal 2004. Our interest expense on deposits decreased by 5.7% from Rs. 41.63 billion in fiscal 2003 to Rs. 39.26 billion in fiscal 2004 as a result of the decrease in interest rates, offset in part by an increase in volume of deposits. Our average total deposits increased by 15.9% from Rs. 675.75 billion in fiscal 2003 to Rs. 783.43 billion in fiscal 2004. Our average cost of deposits declined from 6.2% in fiscal 2003 to 5.0% in fiscal 2004, reflecting primarily the decline in interest rates and a slight increase in the share of low-cost deposits in our deposit mix. Our other interest expense, which consists mainly of Tier 2 bonds issued by us, increased by 12.6% from Rs. 1.91 billion in fiscal 2003 to Rs. 2.15 billion in fiscal 2004 as a result of the issuance in fiscal 2004 of two new series of Tier 2 bonds in an aggregate principal amount of Rs. 7.65 billion. Our average cost of borrowings (including our Tier 2 bonds) declined from 11.2% in fiscal 2003 to 8.2% in fiscal 2004 due to declining interest rates. 100 Other Income Our other income increased by 49.3% from Rs. 12.50 billion in fiscal 2003 to Rs. 18.67 billion in fiscal 2004. The following table sets forth the components of our other income: (In Rs. million) Year ended March 31, 2003 2004 Commission, exchange and brokerage 4,800.1 5,519.0 Profit on sale of investments (net) 6,722.8 12,363.7 Profit (loss) on revaluation of investments (net) (700.8) (1,178.0) Profit on sale of land, buildings and other assets‘ 4.1 2.4 Profit on exchange transactions (net) 950.8 1,060.2 Dividends from subsidiaries, companies and joint ventures 237.0 303.0 Miscellaneous income 489.1 598.5 Total other income 12,503.1 18,668.8 Income from commissions, exchange and brokerage increased by 15% from Rs. 4.80 billion in fiscal 2003 to Rs. 5.52 billion in fiscal 2004. The following table sets forth the components of income from commission, exchange and brokerage: (In Rs. million) Year ended March 31, 2003 2004 Remittances 1,055.3 1,077.6 Documentary credits and guarantees 1,004.6 1,102.2 Bills 925.3 949.6 Commissions on handling Government business such as tax collection and pension disbursements‘ 407.0 479.9 Rent on safe deposit vaults 333.8 340.8 Service charges and processing fees 762.8 1,007.5 Incidental charges on deposit accounts 283.7 518.2 Others 27.6 43.2 Total 4,800.1 5,519.0 Income from commissions, exchange and brokerage increased mainly because of a 32.1% increase in service charges and processing fees from Rs. 0.76 billion in fiscal 2003 to Rs. 1.01 billion in fiscal 2004 as a result of increased services to corporate and retail customers, and an 83.0% increase in incidental charges on deposit accounts from Rs. 0.28 billion in fiscal 2003 to Rs. 0.52 billion in fiscal 2004. Net profit on the sale of investments increased by 83.9% from Rs. 6.72 billion in fiscal 2003 to Rs. 12.36 billion in fiscal 2004. Part of this increase was due to a gain in fiscal 2004 of Rs. 2.93 billion from the repurchase by the Government of some of its outstanding high-coupon securities. The Government offered to repurchase these securities from public sector banks at a 101 discount to the market price by issuing new lower-coupon securities, and permitted the banks to use the net gain tax free to increase their provisioning for non-performing assets. The rest of the increase was primarily due to gains from the higher volume of trading activities we undertook. Our trading profits in future periods may be adversely affected by interest rate fluctuations, particularly in an environment of rising interest rates. Revaluation of investments resulted in a net loss of Rs. 1.18 billion in fiscal 2004 compared to a net loss of Rs. 0.70 billion in fiscal 2003. We account for the amortization of premiums paid to acquire “Held to Maturity” securities under this item, and the increased loss was due to amortization of premiums paid to acquire high coupon “Held to Maturity” securities and mark to market losses of our other securities. Net profit from foreign exchange transactions increased by 11.6% from Rs. 0.95 billion in fiscal 2003 to Rs. 1.06 billion in fiscal 2004, primarily due to a higher volume of foreign exchange trading activities. Dividend income from our subsidiaries and joint ventures increased by 25% from Rs. 0.24 billion in fiscal 2003 to Rs. 0.30 billion in fiscal 2004, primarily because of higher profits earned by these entities in fiscal 2004. Operating Expenses Total operating expenses increased by 15.3% from Rs. 20.57 billion in fiscal 2003 to Rs. 23.71 billion in fiscal 2004. As a percentage of our total income, operating expenses increased to 24.6% in fiscal 2004 compared to 23.5% in fiscal 2003. The primary component of our operating expenses was wages and other payments to employees which increased by 12.1% from Rs. 14.76 billion in fiscal 2003 to Rs. 16.54 billion in fiscal 2004. As a percentage of total income, wages and other payments to employees increased to 17.1% in fiscal 2004 from 16.9% in fiscal 2003. The increase was due to annual increases in remuneration of 6% to 8% and increased contribution towards retirement benefit funds on the basis of actuarial valuations. Our headcount remained substantially unchanged at 58,839 employees as of March 31, 2004 from 58,981 employees as of March 31, 2003. Our expenses for rent, taxes and lighting for our premises increased by 13.2% from Rs. 1.06 billion in fiscal 2003 to Rs. 1.20 billion in fiscal 2004. As a percentage of total income, these expenses were 1.2% in fiscal 2003 and fiscal 2004. Depreciation expenses on our properties increased by 40.3% from Rs. 1.29 billion in fiscal 2003 to Rs. 1.81 billion in fiscal 2004. As a percentage of total revenues, these expenses increased from 1.5% in fiscal 2003 to 1.9% in fiscal 2004. The increase was primarily due to the amortization of goodwill relating to our acquisition of Nedungadi Bank. We amortized Rs. 0.12 billion of this goodwill in fiscal 2003 and wrote off the entire remaining amount of Rs. 0.37 billion in fiscal 2004. As part of the restatement of our financial statements in connection with this Issue, the Rs. 0.37 billion write-off was reversed for fiscal 2004 and allocated to fiscal 2003, as explained below. The increase in our depreciation expenses in fiscal 2004 was also due to our increased investments in technology, consisting primarily of computer hardware and software. We depreciate computer hardware at a rate of 33.3% per year. Gross Profit For the reasons stated above, our gross profit before provisions and contingencies increased by 34.7% from Rs. 23.17 billion in fiscal 2003 to Rs. 31.21 billion in fiscal 2004. As a percentage of total income, our gross profit increased from 26.5% in fiscal 2003 to 32.3% in fiscal 2004. 102 Provisions and Contingencies The following table sets forth, for the periods indicated, the components of our provisions and contingencies: (In Rs. million) Year ended March 31, 2003 2004 Provision for non-performing assets* 4,729.7 4,010.4 Floating provision against non-performing assets 3,600.0 7,930.0 Provision for restructured standard accounts 771.1 1,207.4 Provision for standard accounts 140.0 465.0 Depreciation on investments 1,327.5 (308.6) Provision for income tax 3,406.6 6,599.9 Others 776.1 217.6 Total provisions and contingencies made during the fiscal year 14,751.0 20,121.7 * Net of recovery in written-off accounts of Rs. 1.02 billion in fiscal 2003 and Rs. 2.07 billion in fiscal 2004. Provisions and contingencies made in fiscal 2004 increased by 36.4% to Rs. 20.12 billion compared to Rs. 14.75 billion in fiscal 2003. Our provisioning in respect of non-performing assets (which is the sum of the provision for non-performing assets and the floating provision against non-performing assets in the table above) increased by 43% from Rs. 8.33 billion in fiscal 2003 to Rs. 11.94 billion in fiscal 2004. This included an increase of 120.3% in float provisioning against non-performing assets from Rs. 3.60 billion in fiscal 2003 to Rs. 7.93 billion in fiscal 2004. Of this amount, Rs. 2.93 billion represented our gains from the sale of securities back to the Government, the proceeds of which were used for provisioning. The increase in float provisioning was partly offset by a 15.2% decline in our other provisioning for non-performing assets because of the non-performing assets we wrote off. In addition, our provisioning for restructured standard accounts increased by 56.6% because the implementation of the new corporate debt restructuring mechanism led to an increase in our restructured accounts. Our provisioning for standard accounts increased by 236% from Rs. 0.14 billion in fiscal 2003 to Rs. 0.47 billion in fiscal 2004, mainly as a result of a Rs. 0.30 billion provision for one of our standard accounts and an increase in provisioning to meet the RBI’s requirement to maintain a 0.25% provision for standard accounts. These increases were partly offset by a release in fiscal 2004 of Rs. 0.31 billion of depreciation on investments previously booked compared to a Rs. 1.33 billion depreciation on investments in fiscal 2003. Income Tax Our provision for income tax increased by 93.5% from Rs. 3.41 billion in fiscal 2003 to Rs. 6.60 billion in fiscal 2004. Our tax liability for fiscal 2003 was reduced by Rs. 1.1 billion as a result of tax rulings in our favor in respect of disputed tax amounts for fiscal 2001 and 2002. Further, our tax liability for fiscal 2004 was increased because of provisions of Rs. 0.6 billion that we had to make on account of certain unfavorable tax rulings for earlier years. Without the foregoing adjustments, our provisions for income tax would have increased by 33.9% from fiscal 2003 to fiscal 2004, compared to a 54.4% increase in our taxable income for these periods from Rs. 11.08 billion to Rs. 17.11 billion. Our effective rate of tax was 28.8% in fiscal 2003 and 37.3% in fiscal 2004. Net Profit As a result of the foregoing factors, our net profit increased by 31.7% from Rs. 8.42 billion in fiscal 2003 to Rs. 11.09 billion in fiscal 2004. As a percentage of total income, our net profit increased from 9.6% in fiscal 2003 to 11.5% in fiscal 2004. Our earnings per equity share increased by 30.3% from Rs. 32.08 per equity share in fiscal 2003 to Rs. 41.79 per equity share in fiscal 2004. 103 Adjusted Net Profit Our adjusted net profit for fiscal 2003 was Rs. 7.89 billion, which is Rs. 0.53 billion lower than our previously stated net profit of Rs. 8.42 billion and our adjusted net profit for fiscal 2004 was Rs. 10.61 billion, which is Rs. 0.48 billion lower than our previously stated net profit of Rs. 11.09 billion. In our restated financial statements, we have allocated to fiscal 2003 a depreciation provision of Rs. 0.37 billion which we made in fiscal 2004. This depreciation provision represented the amount of goodwill relating to our acquisition of Nedungadi Bank which we wrote off in fiscal 2004. In addition, as discussed above, we have allocated Rs. 0.46 billion to fiscal 2003 and Rs. 1.10 billion to fiscal 2004 on account of the provisions made in fiscal 2005 relating to the upcoming wage settlement. These allocations and their tax impact resulted in the net downward adjustments to our net profit for fiscal 2003 and fiscal 2004. Fiscal Year Ended March 31, 2003 Compared to the Fiscal Year Ended March 31, 2002 Our total income increased by 14.5% from Rs. 76.26 billion in fiscal 2002 to Rs. 87.35 billion in fiscal 2003 and our total expenditure increased by 4.3% from Rs. 61.52 billion in fiscal 2002 to Rs. 64.18 billion in fiscal 2003. Our gross profit increased by 57.2% from Rs. 14.74 billion in fiscal 2002 to Rs. 23.17 billion in fiscal 2003 and our unadjusted net profit increased by 49.8% from Rs. 5.62 billion in fiscal 2002 to Rs. 8.42 billion in fiscal 2003. We acquired Nedungadi Bank in fiscal 2003 and its results were included in ours for two months in that fiscal year. In fiscal 2002, Nedungadi Bank had total income of Rs. 2.26 billion compared to our total income of Rs. 76.26 billion and as of March 31, 2002, it had total assets of Rs. 15.77 billion compared to our total assets of Rs. 729.15 billion. Net Interest Income Our net interest income increased by 36% from Rs. 22.95 billion in fiscal 2002 to Rs. 31.24 billion in fiscal 2003. The following table sets forth the components of our net interest income: (In Rs. million) Year ended March 31, 2002 2003 Interest income 66,478.7 74,850.1 Interest expense 43,525.8 43,612.9 Net interest income 22,952.9 31,237.2 The increase in net interest income was a result of a 12.6% increase in total interest income from Rs. 66.48 billion in fiscal 2002 to Rs. 74.85 billion in fiscal 2003, and substantially unchanged total interest expense, which was Rs. 43.53 billion in fiscal 2002 and Rs. 43.61 billion in fiscal 2003. This reflected gains in income from advances and income from investments, as well as the growth of our loan portfolio and a decline in interest expense on deposits and borrowings. Our average interest earning assets increased by 15.7% in fiscal 2003 compared to fiscal 2002. Our net interest margin increased from 3.7% in fiscal 2003 to 4.3% in fiscal 2003 and our spread increased from 3.6% in fiscal 2002 to 4.1% in fiscal 2003. 104 Interest Income The following table sets forth the components of our interest income: (In Rs. million) Year ended March 31, 2002 2003 Interest income from advances and bills 33,178.8 37,115.9 Income from investments 30,027.6 32,983.3 Interest on balances with the RBI and other interbanklending 2,276.3 1,793.8 Interest on income tax 163.8 2,152.7 Other interest income 832.2 804.4 Total interest income 66,478.7 74,850.1 Interest income from advances and bills increased by 11.8% from Rs. 33.18 billion in fiscal 2002 to Rs. 37.12 billion in fiscal 2003, reflecting an increase in the average volume of advances as a result of the general growth in our business, offset in part by a decrease in average yield. Our average volume of advances increased by 18.8% from Rs. 310.90 billion in fiscal 2002 to Rs. 369.51 billion in fiscal 2003. Our average volume of advances to corporate and commercial customers increased by 10.2% from Rs. 239.66 billion in fiscal 2002 to Rs. 264.14 billion in fiscal 2003. This included small scale industry advances, which increased by 9.5% from Rs. 38.77 billion in fiscal 2002 to Rs. 42.46 billion in fiscal 2003. These increases reflected a general growth in business with these customer segments. Our average retail advances increased by 66.3% from Rs. 31.96 billion in fiscal 2002 to Rs. 53.14 billion in fiscal 2003 primarily because lower interest rates made retail loans more attractive to customers, particularly in the housing and consumer goods segment, in line with trends experienced by the banking industry as a whole, combined with increased marketing efforts on our part. Our average volume of agriculture advances increased by 32.9% from Rs. 39.29 billion in fiscal 2002 to Rs. 52.23 billion in fiscal 2003 due to our efforts to increase business in this sector. Yields on our advances decreased from an average of 10.7% in fiscal 2002 to 10.0% in fiscal 2003. This decline was due to the general decline in interest rates and increased competition in the banking industry. Investment income increased by 9.8% from Rs. 30.03 billion in fiscal 2002 to Rs. 32.98 billion in fiscal 2003. Our average volume of investments increased by 15.7% from Rs. 274.6 billion in fiscal 2002 to Rs. 317.8 billion in fiscal 2003 because the increase in our deposits provided us with an increased amount of funds to invest. Income from investments in government and other approved securities increased by 13.4% from Rs. 23.7 billion in fiscal 2002 to Rs. 26.9 billion in fiscal 2003. The increase was primarily as a result of a 15.3% increase in the average volume of government securities in our portfolio from Rs. 215.34 billion in fiscal 2002 to Rs. 248.34 billion in fiscal 2003, offset in part by a decline in yields. Yields from government securities declined from 11.0% in fiscal 2002 to 10.8% in fiscal 2003, reflecting the general decline in interest rates during this period. Investment income from investments other than government securities decreased by 3.4% from Rs. 6.4 billion in fiscal 2002 to Rs. 6.1 billion in fiscal 2003 because the decline in yields outweighed the increase in the volume of investments. Yields from these investments declined from 10.7% in fiscal 2002 to 8.8% in fiscal 2003, reflecting the general decline in interest rates during this period. Interest on balances with the RBI and other interbank lending decreased from Rs. 2.28 billion in fiscal 2002 to Rs. 1.79 billion in fiscal 2003, as a result of the lower cash reserve ratio balances we were required to maintain with the RBI and lower interbank lending during the year. Interest on income tax increased significantly from Rs. 0.16 billion in fiscal 2002 to Rs. 2.15 billion in fiscal 2003 because in fiscal 2003 we received a substantial refund of disputed income tax payments attributable to prior years, together with the interest thereon. Interest Expense Our interest expense remained substantially unchanged at Rs. 43.61 billion in fiscal 2003 compared to Rs. 43.52 billion in fiscal 2002. Our weighted average cost of funds was 7.1% in fiscal 2002 and 6.3% in fiscal 2003. 105 Our interest expense on deposits remained substantially unchanged at Rs. 41.63 billion in fiscal 2003 compared to Rs. 41.22 bil lion in fiscal 2002 even though deposits increased, as a result of the decline in interest rates. Our average total deposits increased by 13.8% from Rs. 594.06 billion in fiscal 2002 to Rs. 675.75 billion in fiscal 2003. Our average cost of deposits declined from 6.9% in fiscal 2002 to 6.2% in fiscal 2003. Our interest expense on borrowings from the RBI and other interbank borrowings decreased from Rs. 0.48 billion in fiscal 2002 to Rs. 0.075 billion in fiscal 2003 because of reduced dependence on interbank borrowings. Our other interest expense increased by 4.4% from Rs. 1.83 billion in fiscal 2002 to Rs. 1.91 billion in fiscal 2003 as a result of interest payments on newly issued Tier 2 bonds and interest payments on Rs. 0.25 billion in bonds assumed as part of our acquisition of Nedungadi Bank. Our average cost of borrowings (including our Tier 2 bonds) declined from 13.6% in fiscal 2003 to 11.2% in fiscal 2004. Other Income Our other income increased by 27.9% from Rs. 9.78 billion in fiscal 2002 to Rs. 12.50 billion in fiscal 2003. The following table sets forth the components of our other income: (In Rs. million) Year ended March 31, 2002 2003 Commission, exchange and brokerage 4,339.9 4,800.1 Profit on sale of investments (net) 4,379.1 6,722.8 Profit (loss) on revaluation of investments (net) (581.7) (700.8) Profit on sale of land, buildings and other assets 3.7 4.1 Profit on exchange transactions (net) 922.4 950.8 Dividends from subsidiaries, companies and joint ventures 280.0 237.0 Miscellaneous income 433.8 489.1 Total other income 9,777.2 12,503.1 Income from commissions, exchange and brokerage increased by 10.6% from Rs. 4.34 billion in fiscal 2002 to Rs. 4.80 billion in fiscal 2003. The following table sets forth the components of our income from commission, exchange and brokerage: (In Rs. million) Year ended March 31, 2002 2003 Remittances 1,033.5 1,055.3 Documentary credits and guarantees 909.0 1,004.6 Bills 926.1 925.3 Commissions on handling Government business such as tax collection and pension disbursements ‘ 375.0 407.0 Rent on safe deposit vaults 323.8 333.8 Service charges and processing fees 542.6 762.8 Incidental charges on deposit accounts 178.8 283.7 Others 51.1 27.6 Total 4,339.9 4,800.1 106 Income from commissions, exchange and brokerage increased mainly because of a 40.7% increase in service charges and processing fees from Rs. 0.54 billion in fiscal 2002 to Rs. 0.76 billion in fiscal 2003 as a result of our increased strategic focus from fiscal 2003 onwards on expanding our fee-based services, and an 55.6% increase in incidental charges on deposit accounts from Rs. 0.18 billion in fiscal 2002 to Rs. 0.28 billion in fiscal 2003. Net profit on the sale of investments increased by 53.4% from Rs. 4.38 billion in fiscal 2002 to Rs. 6.72 billion in fiscal 2003. The increase was primarily due to gains from the higher volume of trading activities we undertook to take advantage of interest rate fluctuations. Revaluation of certain investments resulted in a net loss of Rs. 0.70 billion in fiscal 2003 compared to a net loss of Rs. 0.58 billion in fiscal 2002. The increased loss in fiscal 2003 was due to increased amounts attributable to the amortization of premiums paid to acquire high coupon “Held to Maturity” securities. Net profit from foreign exchange transactions increased by 3.3% from Rs. 0.92 billion in fiscal 2002 to Rs. 0.95 billion in fiscal 2003, primarily due to a higher volume of foreign exchange transactions. Dividend income from our subsidiaries and joint ventures decreased by 15.3% from Rs. 0.28 billion in fiscal 2002 to Rs. 0.24 million in fiscal 2003, because in fiscal 2002 we received an interim as well as a regular dividend from our subsidiary PNB Gilts Ltd. Operating Expenses Total operating expenses increased by 14.3% from Rs. 17.99 billion in fiscal 2002 to Rs. 20.57 billion in fiscal 2003. As a percentage of our total income, operating expenses remained substantially unchanged at 23.6% in fiscal 2003 compared to 23.5% in fiscal 2002. The primary component of our operating expenses was wages and other payments to employees which increased by 12.2% from Rs. 13.16 billion in fiscal 2002 to Rs. 14.76 billion in fiscal 2003. The increase was due to annual increases in pay scales of 6% to 8% and the addition of employees of Nedungadi Bank. As a percentage of total income, however, wages and other payments to employees decreased slightly from 17.3% in fiscal 2002 to 16.9% in fiscal 2003. Our headcount increased from 57,859 employees as of March 31, 2002 to 58,981 employees as of March 31, 2003 primarily because of the increased number of employees as a result of our acquisition of Nedungadi Bank. Our expenses for rent, taxes and lighting for our premises increased by 12.7% from Rs. 0.94 billion in fiscal 2002 to Rs. 1.06 billion in fiscal 2003. As a percentage of total income, these expenses were 1.2% in fiscal 2002 and fiscal 2003. Depreciation expenses on our properties increased by 51.8% from Rs. 0.85 billion in fiscal 2002 to Rs. 1.29 billion in fiscal 2003. As a percentage of total revenues, these expenses increased from 1.1% in fiscal 2002 to 1.5% in fiscal 2003. The increase was primarily due to the amortization of goodwill relating to our acquisition of Nedungadi Bank, which amounted to Rs. 124.4 million in fiscal 2003, as well as our increased investments in technology which we depreciate at a rate of 33.3% per annum. Gross Profit For the reasons stated above, our gross profit before provisions and contingencies increased by 57.2% from Rs. 14.74 billion in fiscal 2002 to Rs. 23.17 billion in fiscal 2003. As a percentage of total income, our gross profit increased from 19.3% in fiscal 2002 to 26.5% in fiscal 2003. 107 Provisions and Contingencies The following table sets forth, for the periods indicated, the components of our provisions and contingencies: (In Rs. million) Year ended March 31, 2002 2003 Provision for non-performing assets* 3,913.3 4,729.7 Floating provision against non-performing assets 2,400.0 3,600.0 Provision for restructured standard accounts 155.3 771.1 Provision for standard accounts‘ 150.0 140.0 Depreciation on investments (352.1) 1,327.5 Provisions for income tax 1,981.5 3,406.6 Others 866.1 776.1 Total provisions and contingencies made during the fiscal year 9,114.1 14,751.0 * Net of recovery in written-off accounts of Rs. 0.48 billion in fiscal 2002 and Rs. 1.02 billion in fiscal 2003. Provisions and contingencies made in fiscal 2003 increased by 61.9% to Rs. 14.75 billion compared to Rs. 9.11 billion in fiscal 2002. Our provisioning in respect of non-performing assets increased by 32.0% from Rs. 6.31 billion in fiscal 2002 to Rs. 8.33 billion in fiscal 2003. This included a 50% increase in float provisioning against non-performing assets from Rs. 2.40 billion in fiscal 2002 to Rs. 3.60 billion in fiscal 2003 and a 21.0% increase in our other provisioning for non-performing assets from Rs. 3.91 billion in fiscal 2003 to Rs. 4.73 billion in fiscal 2004. In addition, our provisioning for restructured standard accounts increased significantly from Rs. 0.16 billion in fiscal 2002 to Rs. 0.77 billion in fiscal 2003 because of increased proceedings under India’s new corporate debt restructuring mechanism. Our provisioning for depreciation on investments was Rs. 1.33 billion in fiscal 2003 compared to a Rs. 0.35 billion release in fiscal 2002 of previously booked depreciation on investments. Part of the depreciation on investments in fiscal 2003 was due to depreciation of Rs. 1.34 billion that we booked upon the restructuring of bonds of IFCI that we held in our investment portfolio. Income Tax Our provision for income tax increased by Rs. 1.98 billion in fiscal 2002 to Rs. 3.41 billion in fiscal 2003 mainly because of the increase in our taxable income from Rs. 5.54 billion in fiscal 2002 to Rs. 11.08 billion in fiscal 2003. Our tax liability for fiscal 2003 was reduced by Rs. 1.1 billion as a result of tax rulings in our favor in respect of disputed tax amounts for fiscal 2001 and 2002. Income tax expenses in fiscal 2002 was reduced by Rs. 0.17 billion on account of the release of deferred tax assets which reduced our tax expenditure. Our effective rate of tax was 26.1% in fiscal 2002 and 28.8% in fiscal 2003. Net Profit As a result of the foregoing factors, our net profit increased by 49.8% from Rs. 5.62 billion in fiscal 2002 to Rs. 8.42 billion in fiscal 2003. As a percentage of total income, our net profit increased from 7.4% in fiscal 2002 to 9.6% in fiscal 2003. Our earnings per equity share increased by 21.1% from Rs. 26.50 per equity share in fiscal 2002 to Rs. 32.08 per equity share in fiscal 2003. Adjusted Net Profit Our adjusted net profit for fiscal 2003 was Rs. 7.89 billion, which is Rs. 0.53 billion lower than our previously stated net profit of Rs. 8.42 billion. In our restated financial statements, we have reallocated to fiscal 2003 a depreciation provision of Rs. 0.37 billion, which we made in fiscal 2004. This depreciation provision represented the amount of goodwill relating to our acquisition of Nedungadi Bank which we wrote off in fiscal 2004. The effect of the reallocation is that the entire goodwill relating to the acquisition of Nedungadi Bank is presented as having been written off in fiscal 2003. In addition, we have allocated Rs. 0.46 108 billion to fiscal 2003 on account of the provisions made in fiscal 2005 relating to the upcoming wage settlement. These allocations and their tax impact resulted in the net downward adjustment to our net profit for fiscal 2003. There was no adjustment to the net profit for fiscal 2002. Liquidity and Capital Resources Our growth over the last three fiscal years has been financed by a combination of cash generated from operations, increases in our customer deposits and borrowings, including our Tier 2 bonds. The following table sets forth our cash flows, as restated, from operating activities, investing activities and financing activities in a condensed format. We have aggregated certain items set forth in the cash flow statements as restated that are part of our financial statements included elsewhere in this Red Herring Prospectus in order to facilitate understanding of significant trends in our business. This information should be read together with the full cash flow statements and notes to our financial statements included elsewhere herein. (In Rs. Million) Year ended March 31, First six months of fiscal 2002 2003 2004 2004 2005 Cash flows from operating activities Net profit after tax 5,623.9 7,894.9 10,611.1 5,277.4 7,909.3 Adjustments for taxes (net of deferred tax) 1,983.5 3,100.4 6,345.0 2,676.0 3,135.1 Non cash adjustments to profit before taxes 9,587.5 15,176.9 18,492.8 11,445.1 8,120.7 Cash adjustments to profit before taxes (consisting primarily of interest on Tier 2 bonds and dividends from subsidiaries) 1,304.0 1,450.8 1,648.9 5.9 (266.0) Operating profit before changes in operating assets and liabilities 18,498.9 27,623.0 37,097.8 19,404.4 18,899.1 Adjustment for net change in operating assets and liabilities Decrease / (Increase) in investments (30,425.5) (59,726.1) (80,597.9) (78,960.8) (37,492.4) Decrease / (Increase) in advances (70,058.1) (64,339.3) (76,046.0) (7,822.2) (47,640.3) Increase / (Decrease) in deposits 79,923.5 116,900.2 121,029.0 43,714.0 66,722.4 Increase / (Decrease) in borrowings (2,646.2) 2,535.9 6,268.9 830.6 6,629.6 Change in other assets, liabilities & provisions (712.9) (4,750.1) 7,375.8 5,529.4 (13,777.9) Net change in operating assets and liabilities (23,919.2) (9,379.4) (21,970.2) (36,708.6) (25,558.6) Cash generated from operations (5,420.3) 18,243.6 15,127.6 (17,304.2) (6,659.5) Tax paid (net of refund ) 2,288.3 (1,234.9) (9,834.7) 506.1 2,817.5 Net cash from operating activities (3,132.0) 17,008.7 5,292.9 (16,798.1) (3,842.0) 109 (In Rs. Million) Year ended March 31, First six months of fiscal 2002 2003 2004 2004 2005 Cash flows from investing activities Investment in subsidiaries/joint ventures/RRBs (10.0) 0.0 0.0 168.9 (1,009.5) Purchase of fixed assets (net of sales) (1,280.2) (2,262.6) (2,048.9) (827.5) (829.7) Dividends received from subsidiaries / joint ventures/RRBs 280.0 273.0 303.0 (183.0) (163.0) Loss of PNB Capital taken over 0.0 0.0 (16.1) (16.1) Net cash used in investing activities (1,010.2) (1,989.6) (1,762.0) (857.7) (2,002.2) Cash flows from financing activities Tier 2 bonds issued, net of redemptions 2,900.2 4,130.0 7,650.0 2,650.0 (150.0) Share application money, net of issue expenses 6,699.7 0.0 0.0 0.0 0.0 Interest paid on Tier 2 bonds (1,529.8) (1,723.8) (1,951.9) (891.2) (1,102.4) Payments of dividends / corporate tax on dividends (636.7) (636.7) (1,795.8) (1,047.5) (533.9) Net cash from financing activities 7,433.4 1,769.5 3,902.3 711.3 (1,786.3) Net change in cash and cash equivalents 3,291.2 16,788.6 7,433.2 (16,944.5) (7,630.5) Cash and cash equivalents at the beginning of the year 60,692.1 63,983.3 80,771.9 80,771.9 88,205.1 Cash and cash equivalents at the end of the year 63,983.3 80,771.9 88,205.1 63,827.4 80,574.6 Cash flows from operations Our net cash from operating activities reflects our net profit, adjustments for provision for tax and non-cash charges such as depreciation, amortization and provisions (mainly for non-performing and standard assets) made during the period, as well as adjustments for cash charges consisting primarily of interest on our Tier 2 bonds (which is included in financing activity) and dividends from subsidiaries (which is included in investing activity). In addition, our net cash from operating activities reflects changes in operating assets and liabilities, including investments, advances, deposits and borrowings, as well as other assets and liabilities and provisions. Change in borrowings reflects only short term borrowings and not Tier 2 bonds which are included in financing activity. Our net cash from operating activities fluctuated significantly in the periods shown and was negative in fiscal 2002 as well as in the first six months of fiscal 2004 and 2005, principally because of changes in our operating assets and liabilities. Our profit after tax increased in all the periods shown, as did our operating profit before changes in operating assets and liabilities, which is net of the adjustments for provision for tax as well as noncash and cash adjustments. However, our operating assets increased by more than our operating liabilities in all the periods, which thereby decreased our net cash from operating activities. In particular, in the first six months of fiscal 2004 our cash flow from operating activities was negative because the increases in our advances and investments significantly exceeded the increase in our deposits and borrowings by Rs. 42.24 billion, and there was a Rs. 5.99 billion increase in other assets, which was partly offset by an increase in other liabilities and provisions of Rs. 11.52 billion. In the first six months of fiscal 2005, our cash flow from operating activities was negative because the increases in our advances and investments exceeded the increase in our deposits and borrowings by Rs. 11.38 billion, other assets increased by Rs. 5.96 billion and other liabilities and provisions decreased by Rs. 7.82 billion. 110 Cash flows from investing activities Our net cash used in investing activities was Rs 1.01 billion, Rs 1.99 billion and Rs 1.76 billion in fiscal 2002, 2003 and 2004, respectively, and Rs 0.9 billion and Rs 2.00 billion in the first six months of fiscal 2004 and 2005, respectively. Our net cash used in investing activities reflects principally investments consisting of the purchase of fixed assets, offset in part by dividends received from our subsidiaries. In the first six months of fiscal 2005, we made an investment of Rs. 1.01 billion in certain joint ventures and investment funds. Cash flows from financing activities Our net cash from financing activities was Rs. 7.43 billion, Rs. 1.77 billion and Rs. 3.90 billion in fiscal 2002, 2003 and 2004, respectively, and Rs. 0.7 billion and a negative Rs 1.79 billion in the first six months of fiscal 2004 and 2005, respectively. Our net cash from financing activities reflects principally cash received from the issuance of Tier 2 bonds, which we issue for capital adequacy purposes, issued in each fiscal year shown as well as in the first six months of 2004. We did not issue Tier 2 bonds in the first six months of 2005. In addition, in fiscal 2002 we received Rs. 6.70 billion in share application money from our initial public offering, net of issue expenses, of which we retained Rs. 1.64 billion as issue proceeds. In fiscal 2002 we redeemed Rs. 1.90 billion of Tier 2 bonds at maturity. We also use cash in financing activities to pay interest on Tier 2 bonds and to pay dividends and corporate tax related to dividends. Our dividends increased significantly from fiscal 2003 to fiscal 2004 because we increased our dividend rate and also because we paid an interim dividend. Capital We are subject to the capital adequacy requirements of the RBI, which are primarily based on the capital adequacy accord reached by the Basel Committee of the Bank of International Settlements in 1988. We are required to maintain a minimum ratio of total capital to risk adjusted assets as determined by a specified formula of 9.0%, at least half of which must be Tier 1 capital. Our regulatory capital and capital adequacy ratios based on our restated financial statements are as follows: (In Rs. million) As of March 31, 2003 March 31, 2004 September 30, 2004 Tier 1 capital 28,760.1 32,923.5 40,045.3 Tier 2 capital 20,179.4 29,949.5 28,144.5 Total capital 48,939.5 62,873.0 68,189.8 Total risk weighted assets and contingents 410,899.4 491,729.5 547,531.0 Capital adequacy ratios: Tier 1 7.00% 6.70% 7.31% Tier 2 4.91% 6.09% 5.14% Total capital ratio 11.91% 12.79% 12.45% Minimum capital ratios required by the RBI: Tier 1 4.5% 4.5% 4.5% Total capital ratio 9.0% 9.0% 9.0% As shown above, our Tier 2 capital ratio decreased to 5.14% and our total capital ratio decreased to 12.45% as of September 30, 2004, compared to 6.09% and 12.79%, respectively, as of March 31, 2004. The reduction is attributable to the decrease in Tier 2 capital. This decrease is due to the discounting of our Tier 2 bonds in the amount of Rs. 2.57 billion, which is required pursuant to the RBI guidelines applicable to the calculation of capital ratios. In addition, we redeemed Rs. 150 million of our Tier 2 bonds during the first six months of fiscal 2005. Our Tier 2 capital ratio increased to 6.09% and our total capital ratio increased to 12.79% as of March 31, 2004, compared to 4.91% and 11.91%, respectively, as of March 31, 2003. The increase in our Tier 2 111 capital is primarily due to the issuance of Tier 2 bonds in a principal amount of Rs. 7.65 billion and the transfer of Rs. 4.65 billion of our profits to our investment fluctuation reserve. The RBI Tier 1 capital and total capital ratios are expected to change with the implementation of the Basel II standards in late 2006 or early 2007. Under Basel II, there will be three methods for determining the risk weighting of assets for purposes of calculating capital adequacy, consisting of one “standardized” method in which external standards are used, and two methods in which a bank’s internal standards are used. The RBI has said that Indian banks should first use the standardized method but may later permit banks to change to the internal methods. Our capital adequacy ratio as of September 30, 2004 on a historical, unrestated basis was 12.58%. If the Basel II standardized method were applied on a pro forma basis as of September 30, 2004, we would expect our capital adequacy ratio as of that date to be 9.85%. Financial Condition Our net assets, which we define as our total assets less our total liabilities, increased by 25.7% from Rs. 36.51 billion as of March 31, 2003 to Rs. 45.90 billion as of March 31, 2004, and further increased by 17.2% to Rs. 53.81 billion as of September 30, 2004. Assets The following table sets forth the principal components of our assets as of March 31, 2003, March 31, 2004 and September 30, 2004, as restated: (In Rs. million) As of March 31, 2003 March 31, 2004 September 30, 2004 Cash in hand 6,288.1 6,992.1 5,570.0 Balance with the RBI 59,397.1 60,430.7 60,846.4 Balance with banks 14,322.4 13,811.9 14,107.6 Money at call and short notice 764.2 6,970.4 50.6 Investments 340,300.5 421,254.9 460,185.8 Advances 402,281.2 472,247.2 518,705.4 Fixed Assets (net of revaluation reserve) 5,177.9 5,788.0 5,916.6 Other Assets 30,323.7 33,172.9 32,300.5 Total Assets 858,855.1 1,020,668.1 1,097,682.9 Our total assets increased by 18.8% from Rs. 858.86 billion as of March 31, 2003 to Rs. 1,020.67 billion as of March 31, 2004, and further increased by 7.5% to Rs. 1,097.68 billion as of September 30, 2004. The most significant elements of this increase were increases in investments and advances as a result of the general increase in our business activities. Our investments increased by 23.8% from Rs. 340.3 billion as of March 31, 2003 to Rs. 421.25 billion as of March 31, 2004, and further increased by 9.2% to Rs. 460.19 billion as of September 30, 2004. This was primarily due to increases in our government securities and other investments. Of our total investment portfolio as of September 30, 2004, 84.0% consisted of securities issued by the Government of India and state governments, compared to 84.1% as of March 31, 2004 and 81.5% as of March 31, 2003. Our advances increased by 17.4% from Rs. 402.28 billion as of March 31, 2003 to Rs. 472.25 billion as of March 31, 2004, and further increased by 9.8% to Rs. 518.71 billion as of September 30, 2004. The reasons for the increase in our advances are discussed above under “Results of Operations”. Our money at call and short notice increased significantly from Rs. 0.76 billion as of March 31, 2003 to Rs. 6.97 billion as of March 31, 2004. This was primarily due to a reciprocal deposit we maintained with Deutsche Bank to match foreign currency funds obtained from that bank to meet foreign currency demands from our customers. Our money on call and short notice decreased to Rs. 0.05 billion as of September 30, 2004 due to the termination of the reciprocal deposit. 112 Our gross non-performing assets declined from Rs. 49.8 billion as of March 31, 2003 to Rs. 46.70 billion as of March 31, 2004 to Rs. 41.55 billion as of September 30, 2004 or, as a percentage of total advances, from 11.58% to 9.35% to 7.65%. This was due to our increased recovery efforts, upgrading the classification of assets into “performing” categories and selective write-offs of non-performing assets. Our gross non-performing assets in fiscal 2003 included non-performing assets taken over as part of our acquisition of Nedungadi Bank, which amounted to Rs. 5.17 billion as of the date of the acquisition. Our net non-performing assets declined from Rs. 15.27 billion as of March 31, 2003 to Rs. 4.49 billion as of March 31, 2004 to Rs. 1.49 billion as of September 30, 2004, or, as a percentage of total advances, from 3.86% to 0.98% to 0.30%. This was due to higher provisioning for non-performing assets. See the section titled “Selected Statistical Information” on page 72 for a further discussion of our non-performing assets. Liabilities The following table sets forth the principal components of our liabilities as of March 31, 2003, March 31, 2004 and September 30, 2004: (In Rs. million) As of March 31, 2003 March 31, 2004 September 30, 2004 Demand deposits from banks 1,660.2 1,431.8 929.9 Demand deposits from others 97,222.7 97,572.0 96,552.2 Savings deposits 256,478.9 304,226.1 330,151.0 Term deposits from banks 4,847.8 7,178.2 3,512.9 Term deposits from others 397,925.4 468,755.8 514,740.4 Total deposits 758,135.0 879,163.9 945,886.4 Borrowings 6,621.6 12,890.6 19,520.2 Other liabilities and provisions 41,662.9 59,130.2 55,033.6 Subordinated debt 15,928.6 23,578.6 23.428.6 Total liabilities 822,348.1 974,763.3 1,043,868.8 Our total liabilities increased by 18.5% to Rs. 974.76 billion as of March 31, 2004 from Rs. 822.35 billion as of March 31, 2003. This increase consisted mainly of an 18.6% increase in savings deposits, a 48.1% increase in term deposits from banks, a 17.8% increase in term deposits from others, a 94.7% increase in borrowings, a 39.7% increase in other liabilities and provisions and a 48.0% increase in subordinated debt. The increase in our deposits was principally due to general growth in our business, new customers acquired as we expanded our branch network and greater penetration of our customer base achieved through cross sales of our products. The increase in borrowings was mainly as a result of an increase in foreign currency borrowings. Other liabilities include bills payable, interest accrued on deposits and borrowings and interoffice adjustments. Bills payable increased by 47.3% from Rs. 9.48 billion in fiscal 2003 to Rs. 13.96 billion in fiscal 2004. Our total provisions in respect of non- performing assets increased by 28.8% from Rs. 28.35 billion as of March 31, 2003 to Rs. 36.52 billion as of March 31, 2004. The increase in subordinated debt was largely due to the issuance of Rs. 7.65 billion in Tier 2 bonds in fiscal 2004. Our total liabilities increased by 7.1% from Rs. 974.76 billion as of March 31, 2004 to Rs. 1,043.87 billion as of September 30 , 2004. This increase consisted mainly of a 7.6% increase in deposits as a result of the growth in our business and a 51.4% increase in borrowings as a result of a US$191 million line of credit and external commercial borrowings of US$100 million, which were raised during this period. 113 Off-Balance Sheet Arrangements and Financial Instruments Contingent Liabilities The following table sets forth the principal components of our contingent liabilities as of March 31, 2003, March 31, 2004 and September 30, 2004. (In Rs. million) As of March 31, 2003 March 31, 2004 September 30, 2004 Contingent liabilities Claims against the Bank not acknowledged as debt 1,811.5 2,146.8 2,130.8 Disputed income tax demands under appeal, references, etc. 141.9 377.5 375.7 Liability for partly paid investments 3.4 1.8 1.8 Liability on account of outstanding forward exchange contracts 163,640.3 195,434.7 224,095.9 Guarantees given on behalf of constituents in India and outside India 35,325.4 51,265.2 59,447.7 Acceptances, endorsements and other obligations 33,764.3 71,679.4 90,870.4 Other items for which the Bank is contingently liable 1,021.8 1,394.9 2,553.8 Total 235,708.6 322,300.3 379,476.1 Contingent liabilities increased by 36.7% from Rs. 235.7 billion as of March 31, 2003 to Rs. 322.3 billion as of March 31, 2004 and further increased by 17.7% to Rs. 379.5 billion as of September 30, 2004, primarily due to the following: ● A 19.4% increase in liability on account of forward exchange contracts from Rs. 163.6 billion as of March 31, 2003 to Rs. 195.4 billion as of March 31, 2004 and a further 14.7% increase to Rs. 224.1 billion as of September 30, 2004. This was mainly due to the growth of our foreign exchange business. ● A 45.1% increase in guarantees from Rs. 35.3 billion as of March 31, 2003 to Rs. 51.3 billion as of March 31, 2004 and a further 15.8% increase to Rs. 59.4 billion as of September 30, 2004, because of increased focus on our fee-based services business. ● A 112.3% increase in acceptances, endorsements and other obligations from Rs. 33.8 billion as of March 31, 2003 to Rs. 71.7 billion as of March 31, 2004 and a further 26.8% increase to Rs. 90.9 billion as of September 30, 2004, because of increased focus on our fee-based services business. Foreign Exchange and Derivative Transactions We enter into foreign exchange and derivative transactions for our customers and for our own account. Our foreign exchange contracts arise out of spot and forward foreign exchange transactions with corporate and non-corporate customers and inter- bank counterparties. Our derivative contracts include rupee-based derivatives and cross-currency derivatives for corporate customers and for our own hedging activities. We earn profit on interbank and customer transactions by way of a spread between the purchase rate and the sale rate. Income from foreign exchange transactions is recorded as income from exchange transactions and income from derivatives transactions is recorded as interest income under the head investment income. We use ISDA master agreements for our derivatives transactions. The aggregate notional principal amount of our forward foreign exchange contracts was Rs. 163.64 billion as of March 31, 2003, Rs. 195.43 billion as of March 31, 2004 and Rs. 224.10 billion as of September 30, 2004. Since these contracts are marked to market, their fair value as of those dates was the same as their notional value. Our notional principal amount of our single currency and cross currency interest rate swap agreements was Rs. 15.45 billion as of March 31, 2004, of which Rs. 0.45 billion represented single currency and Rs. 15.0 billion represented cross currency swaps and was Rs. 57.25 billion as of September 30, 2004, of which Rs. 42.25 billion represented single currency and Rs. 15.0 billion represented cross currency swaps. The losses 114 we could incur if the counterparties of these swaps fail to fulfill their obligations would be Rs. 38.2 million and the expected income from these swaps on an accrual basis is Rs. 94.0 million. We did not have any outstanding swaps as of March 31, 2003. Our hedging activities are carried out in the interbank market, which is a non-exchange informal market. However, these markets generally do not provide price discovery or sufficient data to reliably estimate the fair values of financial instruments. Guarantees and Documentary Credits As a part of our commercial banking activities, we issue guarantees and documentary credits such as letters of credit and guarantees to enhance the credit standing of our customers, for which we charge fees and commissions. The amounts of guarantees and documentary credits outstanding as of March 31, 2003, March 31, 2004 and September 30, 2004 are set out below: (In Rs. million) As of March 31, 2003 March 31, 2004 September 30, 2004 Guarantees: Financial guarantees 12,817.4 23,848.7 27,999.6 Performance and other guarantees 22,508.0 27,416.5 31,448.2 Documentary credits 16,239.4 26,403.6 36,281.7 Total 51,564.8 77,668.8 95,729.5 Loan Sanction Letters As part of our business we issue sanction letters to customers, indicating our intent to provide new loans. These letters generally lapse after six months. The letters are issued branch-wise, and we do not compile data on loan sanctions for our bank as a whole. If we were to make these loans, the interest rates would be dependent on lending rates in effect on the date the loans are made. The sanctions are subject to periodic review. Capital Expenditures Our capital expenditures consist principally of investments in branch, office and residential premises, investments in other fixed assets such as furniture and fixtures and investments in computer software. Capital expenditures on premises were Rs. 0.18 billion, Rs. 0.43 billion and Rs. 0.19 billion, respectively, in fiscal 2002, 2003 and 2004. Capital expenditures on other fixed assets were Rs. 1.11 billion, Rs. 1.29 billion and Rs. 1.68 billion, respectively, in fiscal 2002, 2003 and 2004. Capital expenditures on computer software were Rs. 0.37 billion, Rs. 0.06 billion and Rs. 0.13 billion, respectively, in fiscal 2002, 2003 and 2004. We have a maximum capital expenditure budget of Rs. 3.58 billion for fiscal 2005. 115 Selected Financial Data Comparing Standalone to Consolidated Results and Financial Position The following table presents selected standalone and consolidated financial data of PNB as of and for the fiscal years ended March 31, 2003 and 2004. For ease of comparison and because our consolidated financial statements have not been restated, the data is presented on an unadjusted basis and, in the case of the standalone data presented below, will not match the restated unconsolidated financial statements included in this Red Herring Prospectus. Assets and Liabilities Data (In Rs. billions) PNB Standalone PNB Consolidated As of March 31, As of March 31, 2003 2004 2003 2004 Investments 340.3 421.3 350.9 435.9 Advances 402.3 472.2 408.0 478.6 Other Assets 119.6 129.8 122.2 132.4 Total Assets 862.2 1,023.3 881.1 1,046.9 Capital 2.7 2.7 2.7 2.7 Reserves & Surplus 37.7 47.5 41.3 51.9 Deposits 758.1 879.2 759.5 880.7 Borrowings 6.6 12.8 17.3 26.9 Other Liabilities and Provisions 57.1 81.1 60.3 84.7 Total Liabilities 862.2 1,023.3 881.1 1,046.9 Profit and Loss Data (In Rs. billions) PNB Standalone PNB Consolidated Fiscal 2003 Fiscal 2004 Fiscal 2003 Fiscal 2004 Income Interest earned 74.9 77.8 76.7 79.7 Other Income 12.5 18.7 13.4 19.7 Total Income 87.4 96.5 90.1 99.4 Expenditure Interest expended 43.6 41.6 44.7 42.6 Operating expenses 20.6 23.7 20.7 23.9 Provisions and Contingencies 14.7 20.1 15.5 20.9 Total Expenditure 78.9 85.4 80.9 87.4 Share of earnings / loss in associates and other adjustments ¯ ¯ 0.2 0.0 Net Profit (unadjusted) 8.5 11.1 9.4 12.0 116 RECENT DEVELOPMENTS On January 28, 2005, we published our unaudited financial results for the quarter and nine months ended December 31, 2004. These results, together with the results for the quarter and nine months ended December 31, 2003, have been reproduced in this section. Unlike the financial information discussed in the section titled “Management’s Discussion and Analysis of Financial Condition and Results of Operations”, the data for the quarters and nine months ended December 31, 2003 and 2004 has not been restated and therefore is not directly comparable to that information. The following is a summary discussion of our financial results for the quarter ended December 31, 2004: ● Total income increased by 0.76% from Rs. 23.62 billion in the quarter ended December 31, 2003 to Rs. 23.80 billion in the quarter ended December 31, 2004. Interest income increased by 10.2% from Rs. 19.35 billion in the quarter ended December 31, 2003 to Rs. 21.32 billion in the quarter ended December 31, 2004. This increase consisted of an increase of 15.2% in interest on advances and an increase of 5.5% in interest on investments. These increases were due to a 24.2% increase in the average volume of advances from Rs. 428.15 billion to Rs. 531.69 billion and a 14.4% increase in the average volume of investments from Rs. 411.75 billion to Rs. 471.09 billion, partly offset by a decline in interest rates. Other income decreased by 42% from Rs. 4.28 billion in the quarter ended December 31, 2003 to Rs. 2.48 billion in the quarter ended December 31, 2004, primarily because of lower treasury profits. Interest expense increased by 4.3% from 10.58 billion in the quarter ended December 31, 2003 to Rs. 11.03 billion in the quarter ended December 31, 2004, reflecting primarily an increase of 18.5% in the average volume of our deposits from Rs. 792.66 billion to Rs. 938.94 billion, offset partly by a decline in interest rates. Operating expenses increased by 23.8% from Rs. 5.92 billion in the quarter ended December 31, 2003 to Rs. 7.33 billion in the quarter ended December 31, 2004. This was primarily on account of a Rs. 1.23 billion additional expense for retirement benefits. Total expenses increased by 11.3% from Rs. 16.50 billion in the quarter ended December 31, 2003 to Rs. 18.36 billion in the quarter ended December 31, 2004. Net interest income increased by 17.3% from Rs. 8.77 billion in the quarter ended December 31, 2003 to Rs. 10.29 billion in the quarter ended December 31, 2004. Gross profit before provisions and contingencies decreased by 23.5% from Rs. 7.12 billion in the quarter ended December 31, 2003 to Rs. 5.45 billion in the quarter ended December 31, 2004. Net profit increased by 20.8% from Rs. 2.60 billion in the quarter ended December 31, 2003 to Rs. 3.14 billion in the quarter ended December 31, 2004. Provisions and contingencies decreased by 49.1% from Rs. 4.52 billion in the quarter ended December 31, 2003 to Rs. 2.30 billion in the quarter ended December 31, 2004. In the quarter ended December 31, 2004, we made a Rs. 1.07 billion provision on account of the impending banking industry wage settlement, which is described in the section titled “Management’s Discussion and Analysis of Financial Condition and Results of Operations”. Of this amount, Rs. 0.70 billion related to prior periods. Provisions and contingencies were lower in the quarter ended December 31, 2004 because of lower provisions for non-performing assets as a result of improved recoveries which led to a decrease in our net non- performing assets. Our capital adequacy ratio as of December 31, 2004 was 13.11%, which reflected in part the increased risk weights on consumer credit and investments in mortgage backed securities pursuant to new directives implemented by the RBI on December 23, 2004. On January 28, 2005, our board of directors approved an interim dividend of Rs. 3 per Equity Share, which will result in a total payout of Rs. 898 million (of which Rs. 102 million represents dividend tax). The record date for the interim dividend is February 28, 2005 and the dividend will be paid on March 7, 2005. 117 The following is a reproduction of the financial results that were taken on record by our Board of Directors on January 28, 2005 and filed with the Stock Exchanges. One crore represents ten million. FINANCIAL RESULTS ( REVIEWED) FOR THE QUARTER/NINE MONTHS ENDED 31st DECEMBER , 2004 Rs. In Crore Quarter Ended Nine Months Ended Year Ended S.No. Particulars 31.12.2004 31.12.2003 31.12.2004 31.12.2003 31.03.2004 (Reviewed) (Reviewed) (Reviewed) (Reviewed) (Audited) 1 Interest Earned ( a+b+c+d ) 2132.08 1934.59 6275.20 5751.48 7779.69 a) Interest/discount on advances/ bills 1093.05 948.71 3166.29 2880.70 3876.01 b) Income on Investments 981.25 929.82 2951.89 2710.91 3680.97 c) Interest on Balances with RBI & other Inter Bank Funds 43.64 11.86 109.25 68.05 113.13 d) Others 14.14 44.20 47.77 91.82 109.58 2 Other Income 248.00 427.74 1298.33 1476.77 1866.88 A. TOTAL INCOME (1+2) 2380.08 2362.33 7573.53 7228.25 9646.57 3 Interest Expended 1102.57 1057.99 3340.54 3143.81 4154.99 4 Operating Expenses (e+f) 733.01 592.46 2052.76 1662.81 2370.72 e) Payments to and provisions for employees 547.24 406.84 1498.80 1167.10 1654.06 f) Other operating expenses 185.77 185.62 553.96 495.71 716.66 B. TOTAL EXPENSES (3+4) 1835.58 1650.45 5393.30 4806.62 6525.71 (excluding provisions & contingencies) C. Operating Profit (A-B) 544.50 711.88 2180.23 2421.63 3120.86 (Profit before Provisions & Contingencies) Total Provision & Contingencies 230.23 452.08 1130.74 1610.31 2012.17 D. Other Provisions & Contingencies 132.38 227.08 749.77 1104.60 1351.38 - of which Provision for Non-Performing Assets 97.35 197.74 231.18 991.26 1401.00 E. Provision For Taxes 97.85 225.00 380.97 505.71 660.79 F. NET PROFIT (C-D-E) 314.27 259.80 1049.49 811.32 1108.69 5 Paid up equity Share Capital 265.30 265.30 265.30 265.30 265.30 6 Reserves excluding revaluation reserves ( As per Balance Sheet of Previous year) 4425.46 7 Analytical Ratios (i) Share holding of Govt. of India (%) 80 80 80 80 80 (ii) Capital Adequacy Ratio (%) 13.11 12.57 13.11 12.57 13.10 (iii) Earning per Share in Rs. (Non-Annualised, at Face Value of Rs.10/-) 11.85 9.79 39.56 30.58 41.79 (iv) (a) Amount of gross non-performing assets 4188.20 5171.85 4188.20 5171.85 4670.13 (b) Amount of net non-performing assets 137.30 883.76 137.30 883.76 448.96 (c) % of gross NPAs 7.80 11.64 7.80 11.64 9.35 (d) % of net NPAs 0.28 2.20 0.28 2.20 0.98 (v) Return on Assets (Annualised) % 1.11 1.09 1.24 1.14 1.08 8 Aggregate of Non-Promoter Share holding (i) No. of Shares 53061200 53061200 53061200 53061200 53061200 (ii) Percentage of Share holding 20 20 20 20 20 118 Segment Reporting: (Rs. in crore) Business Segments Treasury Other banking operations Unallocated Total Particulars Nine Nine FY 04 Nine Nine FY 04 Nine Nine FY 04 Nine Nine FY 04 Months Months Months Months Months Months Months Months Ended Ended Ended Ended Ended Ended Ended Ended 31.12.04 31.12.03 31.12.04 31.12.03 31.12.04 31.12.03 31.12.04 31.12.03 Segment Revenue (external revenue) 3183.41 3817.85 4913.87 4390.12 3410.40 4732.71 XXX XXX XXX 7573.53 7228.25 9646.58 Segment Results (before Prov and Contingencies & I.Tax) 717.17 1489.07 1912.30 1674.78 1129.62 1501.94 XXX XXX XXX 2391.95 2618.69 3414.24 Provisions & Contingencies XXX XXX XXX XXX XXX XXX XXX XXX XXX 750.38 1104.60 1351.38 Unallocated Corporate Expenses XXX XXX XXX XXX XXX XXX 211.72 197.06 294.18 211.72 197.06 294.18 Income tax XXX XXX XXX XXX XXX XXX XXX XXX XXX 380.36 505.71 659.99 Net Profit XXX XXX XXX XXX XXX XXX XXX XXX XXX 1049.49 811.32 1108.69 Capital Employed 2370.14 3153.30 2111.76 3065.95 1446.40 2834.22 529.00 161.77 65.82 5965.09 161.77 5011.80 Since the transactions of the newly opened Kabul branch are less than the threshold limit of 10%, the same has not been considered as a separate reportable segment. Notes: i. The above financial results were taken on record by the Board of Directors in its meeting held on 28.01.2005. The results were subjected to limited review by the Statutory Central Auditors. ii. The results have been prepared on the basis of the Accounting policies as were followed in the immediate preceding financial year 2003-04. iii. Interim dividend @ 30% amounting to an appropriation of Rs.89.79 Crore (including dividend Tax of Rs.10.20 crore), was approved by Board of Directors of the Bank. iv. The Bank has built up an over and above additional provision of Rs.246 Crores (during the quarter Rs.123 Crore) against pension and gratuity. v. Results for the nine months period ended 31.12.2004 include Rs.207.70 Crores on account of depreciation on certain Govt. securities transferred from AFS to HTM category in terms of RBI guidelines. The figures of the corresponding period are not comparable to that extent. vi. Results for the nine months period ended 31.12.2004 include Miscellaneous Income on account of the credit balance to the tune of Rs.387.07 Crore transferred from Inter branch Transactions blocked account. The said Income has been appropriated to Rs.290.30 Crore in Revenue & Other Reserves and Rs.96.77 Crore to Statutory Reserves. The accounting effect of the above has been given as per approval of RBI. vii. Financial results for the quarter/nine months period ended 31.12.2004 have been arrived at after considering provisions for loan losses and Standard Assets, depreciation on Investments and Fixed assets, Gratuity, Pension, Bonus, Leave Encashment, Tax on Income and amortisation of deferred expenditure on VRS-2000. viii. Provision and contingencies include an adhoc provision of Rs.265 Crore during the nine months period ended 31st December 2004 (Rs.106.75 Crore during the quarter) towards arrear of salary payable to employees, pending settlement on wage revision, under Industry level negotiations. ix. Any complaints from the Investors were not pending at the beginning of the quarter. During the quarter ended on 31.12.2004, 482 complaints were received and all such complaint stand resolved at the end of the period. x. Figures of the corresponding previous period have been regrouped/rearranged/reclassified wherever considered necessary. xi. Aforesaid notes annexed to and form part of the financial results. Place: New Delhi Dr. K.C Chakraborty S.S. Kohli Date: 28.01.2005 Executive Director Chairman & Managing Director 119 OUR MANAGEMENT Board of Directors Under our Constitutional Documents we can have a maximum of 15 Directors. We currently have 9 Directors on our Board of Directors. The following table sets forth details regarding our Board of Directors: Name, Designation, Age Address Other Directorships Occupation and Term Mr. S. S. Kohli, 59 20, Rajdoot Marg, ● Agricultural Finance Corporation Ltd. Chairman & Managing Director. years Chanakaya Puri, ● Oriental Insurance Company Ltd. Whole time Director. New Delhi 110 021. ● PNB Gilts Ltd. (Chairman) Tenure ends on April 19, 2005. ● PNB Housing Finance Ltd. (Chairman) ● Small Industries Development Bank of India. ● Export Credit and Guarantee Corporation of India Ltd. Dr. K.C. Chakrabarty, 52 8, Major Dhyan Chand Marg, ● PNB Gilts Ltd Executive Director. years Asiad Village, ● PNB Housing Finance Limited Whole time Director. New Delhi 110 049. ● PNB Assets Management Company Ltd. Tenure ends on ● Assets Care Enterprises Limited. August 26, 2009. Dr. K.V. Rajan, 54 A-26, RBI Staff Quarters, Nil. RBI Nominee Director. years Char Imli, Bhopal 462 016, Tenure will end on further Madhya Pradesh orders from the MoF. Mr. P. K. Nayar, 55 B-XIX-569, Malerkotla House, Nil. Officer Representative Director. years Civil Lines, Tenure ends on February 19, Ludhiana 141 001, Punjab. 2007 and thereafter until his successor is appointed or till he ceases to be an officer of PNB or until further orders, whichever is earlier, provided that he shall not hold office continuously for a period of six years. Dr. Pritam Singh. 63 F1, Director’s Bungalow, ● Shipping Corporation of India. Shareholder Independent years Management Development ● Indian Credit Rating Agency. Director. Tenure ends on Institute Complex, ● Indian Medicines and Pharmaceuticals Ltd. August 5, 2005. Mehrauli Road, Sukhrali, ● Local Board of Reserve Bank of India. Gurgaon, Haryana ● Oriental Insurance Company Ltd. ● Hindustan Aeronautics Ltd. ● Hero Honda Motors Limited. 120 Name, Designation, Age Address Other Directorships Occupation and Term Dr. K. B. L. Mathur. 58 House no. 9, ● Tourism Finance Corporation of India Economic Advisor, years Upper Ground Floor, Government of India- Director. Road No. 46A, The tenure is till further orders West Punjabi Bagh, of the Goverrnment, or till New Delhi- 110026. he ceases to be an officer of the MoF. Mr. Mohanjit Singh. 70 11, Mathura Road, ● Delfin Finance Ltd. Shareholder Independent years Near Rajdoot Hotel, ● Delhi Financiers (Pvt. Ltd.) Director. Tenure ends on New Delhi 110 014. ● The New India Industrial Corporation Ltd. August 5, 2005. ● Hexa Estates (P) Ltd. ● PNB Gilts Ltd. ● National Institute of Fashion Technology (Members of Board of Governors). Mr. A.S.Agarwal. 59 C/o Swarup Chemicals (P) Ltd. ● Swarup Publications (P) Ltd. Non Executive Independent years Water Works Road. Aishbagh, Director. Tenure ends on Lucknow 226004, November 24, 2006. Uttar Pradesh. Mr. Mohan Lal Bagga., 55 688, Sector 13 Extension, Nil. Workmen Director. years Urban Estate, Karnal, Haryana. Tenure ends on August 31, 2007 and thereafter until his successor is appointed or till he ceases to be a workman employee of the Bank, or until further orders, whichever is earlier provided that he shall not hold office continuously for a period exceeding six years. Note: All directors except for Mr. S.S. Kohli and Dr. K.C. Chakrabarty are non-executive directors. Details of Directors Mr. S. S. Kohli, aged 59 years, the Chairman and Managing Director of the Bank, has a career spanning 34 years in the banking sector. He has degree in mechanical engineering, a diploma in Industrial Finance and CAIIB. He joined the Board of Directors in April 2000. Dr. K. C. Chakrabarty, aged 52 years, Executive Director, has a career spanning 26 years in the banking sector. He has a bachelors degree and a masters degree in science and a doctorate degree in statistics. He joined the Board of Directors in July 2004. Dr. K. V. Rajan, aged 54 years, Director, is presently a Regional Director of the RBI, Bhopal. He has a master’s degree in economics and doctorate degree and has participated in the advanced studies programme of the Kiel Institute of World Economics, Kiel, Germany. He has served in various capacities in various departments of the RBI including the Department of Banking Operations and Development and the Department of Non-Banking Finance Companies. He has joined the Board of Directors in 2004. Mr. P. K. Nayar, aged 55 years, Director, he has a bachelor degree in agricultural science. He has 33 years of experience as an employee in the Bank. He has joined the Board of Directors in 2004. 121 Dr. Pritam Singh, aged 63 years, Director, is a former director of the Indian Institute of Management, Lucknow and is presently the director of Management Development Institute, Gurgaon, Haryana. He has masters in commerce, a masters in business administration and a doctorate degree in management. He has joined the Board of Directors in 2002. Mr. Mohanjit Singh, aged 70 years, Director, is a former director of UCO Bank. He has experience in industry and exports of more than 40 years. He has a bachelor’s degree in economics and law. He has joined the Board of Directors in 2002. Dr. K. B. L. Mathur, aged 58 years, Director, is presently working as Economic Advisor in the Banking Division of the Department of Economic Affairs, MOF, GoI. He has joined the Board of Directors on January 24, 2005. Mr. A. S. Agarwal, aged 59 years, Director, is a businessman and a journalist. He has a bachelor’s degree in commerce and law and a special diploma in taxation. He joined our Board of Directors in 2003. Mr. Mohan Lal Bagga, aged 55 years, Director, has 31 years of experience as an employee in the Bank. He has a bachelor’s degree in science. He has joined the Board of Directors in 2004. Shareholding of our Directors in our Bank Our Constitutional Documents do not require our Directors, except the Directors appointed by shareholders (other than the President of India) to hold any Equity Shares. The shareholding of our Directors as on the date of this Red Herring Prospectus is as follows. Name of the Director Number of Equity Shares of Rs. 10 each Percentage of Outstanding Equity Capital Dr. Pritam Singh 500 Negligible Mr. Mohanjit Singh 100 Negligible There are no transactions by Directors or their relatives in our Equity Shares in the last six months. Term of Office The following table has term of office of our Directors: Name, Status of Director Term Mr. S. S. Kohli, Chairman & Managing Director, Whole Time Director Tenure ends on April 19, 2005. Dr. K.C. Chakrabarty, Executive Director, Whole Time Director Tenure ends on August 26, 2009. Dr. K.V. Rajan, Part time Director, RBI Nominee Tenure will end on further orders from the MoF. Mr. P. K. Nayar, Part time Director, Officer Representative Tenure ends on February 19, 2007. Dr. Pritam Singh, Non Executive Independent Director Tenure ends on August 5, 2005. Dr. K. B. L. Mathur, Government Nominee Director. Tenure is till further orders of the Goverrnment, or till he ceases to be an officer of the MoF. Mr. Mohanjit Singh, Shareholder Independent Director. Tenure ends on August 5, 2005. Mr. A. S. Agarwal, Non Executive Independent Director Tenure ends on November 24, 2006. Mr. Mohan Lal Bagga, Workman Director Tenure ends on August 30, 2007 until his successor is appointed or till he ceases to be a workman employee of the Bank, or until further orders, whichever is earlier provided that he shall not hold office continuously for a period exceeding six years. For details of the terms of appointment of our whole time Directors, please refer to the section titled “Statutory and Other Information” on page 243. 122 Changes in our Board of Directors during the last three years The changes in our Board of Directors during the last three years are as follows: Name Date of Appointment Date of Cessation Reason Mr. V.K. Sibal February 2, 2002 May 7, 2003 Ceased to be Director pursuant to intimation from the GoI. Mr. Ramesh Chander April 2, 2002 July 31, 2003 Ceased to be a Director as per intimation from the GoI. Mr. V.K. Sharma July 31, 2003 January 9, 2004 Ceased to be a Director as per intimation from the GoI. Mr. A. S. Agarwal November 25, 2003 Continuing Appointed as a Director. Dr. K.V. Rajan January 9, 2004 Continuing Nominated as Director by Central Government on recommendation by RBI. Mr. P. K. Nayar February 20, 2004 Continuing Nominated as a Director by the GoI. Mr. T.S. Narayanasami December 15, 2000 April 20, 2004 Ceased to be Director as he was appointed as the Chairman and Managing Director of Andhra Bank by the GoI. Dr. K. C. Chakrabarty August 27, 2004 Continuing Appointed as a Director by the GoI. Mr. M.L. Bagga September 1, 2004 Continuing Nominated as a Director by the GoI. Dr. K. B. L. Mathur January 24, 2005 Continuing Appointed as Director by the GoI. Corporate Governance We had made a public issue of 53,060,700 equity shares in March 2002 and are currently listed on the NSE and the BSE. We had entered into listing agreements with these stock exchanges and we are in compliance with the applicable provisions of the listing agreements with these stock exchanges especially relating to corporate governance, broad basing of management and setting up necessary committees like the Audit Committee and the Shareholders’/ Investors’ Grievance Committee. We have complied with applicable SEBI Guidelines in respect of corporate governance specially with respect to broad basing of board, constituting the committees such as shareholding/ investor grievance committee details of which are provided hereinbelow. We shall comply with the applicable requirements of SEBI circular bearing number SEBI/CFD/DIL/CG/1/2004 dated October 29, 2004, which notifies revised corporate governance guidelines, by the required date, which is April 1, 2005 for listed entities like our Bank. We have constituted the following committees of our Board of Directors for compliance with corporate governance requirements: (a) Audit Committee; (b) Share Transfer Committee (c) Shareholder/Investors Grievance Committee The Board of Directors has not constituted any remuneration committee as the remuneration of Directors is fixed by the Government and guided by guidelines of the Government in this regard. (a) Audit Committee The audit committee comprises one executive and three non-executive Directors, namely, Dr. K.C. Chakrabarty, who is an executive Director, Dr. K. B. L. Mathur, nominee of the Government of India, Dr. K.V. Rajan, who is the nominee of the RBI, Mr. Mohanjit Singh and Mr. A.S. Agarwal, both of whom are non executive independent Directors. We have appointed Ms. Malathi Mohan, Company Secretary as the secretary to the Audit Committee as required under the listing agreements. The 123 responsibilities of the audit committee inter alia include the following: ● Providing direction and overseeing the total audit function of the Bank and follow up on the statutory/external audit of the Bank and inspections of RBI. ● Obtaining and reviewing half yearly reports from the companies cell. ● Interacting with statutory auditors before finalization of annual/semi-annual/quarterly financial accounts and reports and also follow up on all the issues raised in the long form audit report. ● Reviewing the internal inspection/audit functions of the Bank namely the systems, its quality and effectiveness in terms of follow up. ● Reviewing inspection reports of specialized and extra large branches and all the branches with unsatisfactory rating. ● Acting as per provisions of the listing agreements with the Stock Exchanges and in line with the RBI guidelines. (b) Share Transfer Committee This committee approves and monitors share transfers, issue of duplicate share certificates and matters relating thereto. It comprises of Dr. K.C. Chakrabarty, who is also chairman of the committee and an executive Director, Mr. P.K. Nayar, an officer Director, and Mr. Mohanjit Singh, a non-executive Director. (c) Shareholders/Investors Grievance Committee This committee looks into the redressal of shareholders’ grievances. It comprises of Mr. Mohanjit Singh, a non-executive Director, who is also chairman of the committee, Dr. K.C. Chakrabarty, an executive Director and Mr. P. K.Nayar, an officer Director. The Board of Directors has nominated Ms. Malathi Mohan, company secretary, as the compliance officer in terms of Clause 47 of the listing agreements with the Stock Exchanges. Key Managerial Personnel Mr. S. K. Awasthi, aged 59 years, joined our Bank on August 22, 1966 and is currently General Manager. He has a masters degree in commerce and CAIIB and a diploma in bank management. He has 38 years of experience with our Bank. Mr. C. P. Swarnkar, aged 56 years, joined our Bank on December 1, 1969 and is currently General Manager. He has a masters degree in commerce and CAIIB-I. He has 35 years of experience with our Bank. Mr. P. L. Madan, aged 58 years, joined our Bank on July 8, 1967 and is currently General Manager. He has a bachelors degree in commerce, a masters degree in economics and CAIIB. He has 37 years of experience with our Bank. Mr. K. G. Sathyasingan, aged 57 years, joined New Bank of India on July 1, 1975 which was merged with our Bank in 1993, and is currently General Manager. He has a bachelors degree in arts, masters degree in science and CAIIB-I, Diploma in Systems Management, degree in law and diploma in laws relating to air and outer space. He has 29 years of experience with Bank. Mr. V. Nagaraja, aged 57 years, joined our Bank on September 9, 1969 and is currently General Manager. He has a masters degree in commerce and CAIIB-I. He has 35 years of working experience with our Bank. Mr. B. M. Mittal, aged 55 years, joined our Bank on September 1, 1972 and is currently General Manager. He has a bachelor degree in engineering and CAIIB, a post graduate diploma in bank management from National Institute of Bank Management and a post graduate diploma in training and development from Indian Society for Training and Development. He has 32 years of experience with our Bank. Mr. D. L. Rawal, aged 53 years, joined New Bank of India on February 1, 1972 and is currently General Manager. He has a bachelor degree in science and CAIIB. He has 32 years of experience in the banking sector. Mr. A. D. Paliwal, aged 58 years, joined our Bank on March 2, 1971 and is currently General Manager. He has a masters degree in business management and CAIIB besides a post graduate diploma in training and development from Indian Society for Training and Development and diploma in management consultancy from Institute of Management Consultants of India. He has 33 years of experience with our Bank. Mr. U. S. Bhargava, aged 57 years, joined our Bank on March 1, 1971 and is currently General Manager. He has a bachelor degree in technology and CAIIB-I. He has 33 years of experience with our Bank. 124 Mr. A. Balasubramanian, aged 55 years, joined our Bank on December 11, 1981 and is currently General Manager. He has a bachelor degree in science and he is an associate member of the Institute of Chartered Accountants of India. He has 23 years of experience with our Bank. Mr. V.K. Nagar, aged 53 years, joined our Bank on February 26, 1983 and is currently General Manager. He has a bachelor degree in technology and CAIIB, a post graduate diploma in marketing and sales management and a masters degree in business administration. He has 21 years of experience with our Bank. Mr. H. Singh, aged 56 years, joined our Bank on September 1, 1972 and is currently General Manager. He has a masters degree in science (agricultural economics) and CAIIB-I. He has 32 years of experience with our Bank. Mr. A. Kaul, aged 48 years, joined our Bank on July 25, 1983 and is currently General Manager. He has a bachelors degree in science and a masters degree in business administration and CAIIB-I. He has 21 years of experience with our Bank. Mr. R.I.S. Sidhu, aged 54 years, joined our Bank on March 1, 1971 and is currently General Manager. He has a masters degree in economics and CAIIB-I and a post graduate diploma in bank management. He has 33 years of experience with our Bank. Mr. V. P. Choudhary, aged 57 years, joined or Bank on March 1, 1971 and is currently General Manager. He has a bachelors degree in Commerce, masters degree in Economics and CAIIB-I. He has 33 years of experience with our Bank. Mr. L. P. Agarwal, aged 55 years, joined the Bank on December 19, 1981 and is currently General Manager. He has a bachelors degree in commerce, bachelors degree in law and is an associate member of the Institute of Chartered Accountants of India. He has 23 years of experience with our Bank. Mr. Ranjan Dhawan, aged 49 years, joined the Bank on June 27, 1983 and is currently General Manager. He has a bachelors degree in commerce, masters degree in business administration, associate member of the Chartered Institute of Management Accountants (U.K.). He has 21 years of experience with our Bank. Mr. I. D. Singh, aged 54 years, joined the bank on May 20, 1970 and is currently General Manager. He has a Bachelors degree in Commerce and CAIIB. Presently he is the Managing Director of PNB Gilts Ltd., a subsidiary of our Bank. He has around 34 years of experience with our Bank. Mr. P. K. Mitra, aged 55 years, joined the Bank on September 1, 1972 and is currently General Manager. He has a bachelor’s degree in mechanical engineering, CAIIB, post graduate diploma in personnel management and masters in business administration. He has around 32 years of experience with the Bank. Mr. B.P. Chopra, aged 54 years, joined the Bank on September 5, 1973 and is currently General Manager. He has a masters of sciences degree in physics, and CAIIB. He has around 31 years of experience with the Bank. Mr. K. S. Bajwa, aged 56 years, joined the Bank on July 8, 1974 and is currently General Manager. He has a masters degree in arts, masters degree in management, and CAIIB. He has around 30 years of experience with the Bank. None of our key managerial personnel are related to each other. All our key managerial personnel are permanent employees of our Bank. Our key management personnel are entitled to the compensation and benefits are as applicable to our permanent employees. All the Key Management Personnel are of the General Manager grade and hence the compensation falls in the scale of Rs19340-420/2-520-600-21300 per month. 125 The number of shares held by our key management personnel are given below: Name of Key Managerial Personnel No. of Equity Shares held Mr. L.P. Agarwal 100 Mr. P. K. Mitra 200 Mr. B.M. Mittal 100 Mr. R. Dhawan 100 Mr. A.D. Paliwal 400 Mr. Arun Kaul 100 Mr. U.S. Bhargava 100 Mr. V. Nagaraja 100 Mr. V. P. Choudhary 100 Mr. Harwant Singh 200 Mr. R.I.S Sidhu 200 Mr. B.P. Chopra 200 Changes in our key managerial personnel during the last three years The changes in the key managerial personnel in the last three years are as follows: Name Date of Appointment/ Whether continuing, Reason Promotion as Key if not, date of Managerial Personnel cessation Mr. K.N. Prithiviraj June 21, 1996 January 20, 2003 Appointed Executive Director to United Bank of India Mr. D.P. Singh September 30, 1998 March 25, 2004 Death Mr. M. S. Aftab March 28, 2001 November 2, 2004 Appointed as Executive Director to Vijaya Bank Mr. A. Balasubramanian April 16, 2003 Yes Promotion Mr. V.K. Nagar April 16, 2003 Yes Promotion Mr. H. Singh April 16, 2003 Yes Promotion Mr. A. Kaul April 16, 2003 Yes Promotion Mr. R.I.S. Sidhu April 16, 2003 Yes Promotion Mr. V. P. Choudhary May 31, 2004 Yes Promotion Mr. L. P. Agarwal May 31, 2004 Yes Promotion Mr. Ranjan Dhawan May 31, 2004 Yes Promotion Mr. I. D. Singh May 31, 2004 Yes Promotion Mr. P. K. Mitra May 31, 2004 Yes Promotion Mr. B.P. Chopra May 31, 2004 Yes Promotion Mr. K. S. Bajwa May 31, 2004 Yes Promotion 126 OUR PROMOTER, SUBSIDIARIES AND GROUP COMPANIES PROMOTER Our Promoter is the President of India, acting through the MoF, GoI, which holds 80% of the pre-Issue paid up equity share capital of our Bank and will hold at least 67.31% of the fully diluted post-Issue paid up equity share capital of our Bank. Following the Capital Reduction, the President of India will hold 57.80% of the paid up capital of our Bank. For further details on the Capital Reduction please see the section titled “Capital Structure” on page 22. SUBSIDIARIES We have three subsidiaries, namely PNB Housing Finance Limited, PNB Gilts Limited and PNB Asset Management Company Limited. PNB Housing Finance Limited (“PNB Housing”) PNB Housing, a wholly owned subsidiary of our Bank, was incorporated on November 11, 1988. PNB Housing was set up to meet the financing demand from the housing sector. The total amount of loans given by PNB Housing as of December 31, 2004 was Rs. 8794.6 million. Shareholding Pattern The entire share capital is held by the Bank. Board of Directors The directors of PNB Housing are Mr. S.S.Kohli, Dr. K.C. Chakrabarty, Mr. R. S. Awasthi, Mr. Man Mohan Singh Rekhrao, Mr. K. B. Sharma, Mr. Bimal Kumar Raizada, Mr. C. P. Swarnkar, and Mr. K. G. Sathyasingan. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital 300.0 300.0 300.0 Reserve and surplus 376.1 458.1 575.8 Total income 669.2 803.7 904.4 Profit after tax 91.8 119.2 154.9 Earnings per share (Rs.) 3.06 3.97 5.17 Net asset value per share (Rs.) 22.52 25.27 29.19 PNB Gilts Limited (“PNB Gilts”) PNB Gilts, a subsidiary of our Bank, was incorporated on March 13, 1996 and listed in the NSE and BSE. PNB Gilts is a primary dealer appointed by the RBI to undertake market-making activities in Government securities. It is engaged in the business of trading in Government securities, treasury bills, bonds of public sector enterprises, government guaranteed bonds and state government securities. It is also engaged in money market operations, which includes dealing in call/notice and term money, commercial paper and certificate of deposit. The company has also ventured into other areas of financial services, which include merchant banking activities (viz. project appraisals, loan syndication, placement of debts), advisory services, marketing and distribution of mutual fund products, etc. Shareholding Pattern The shareholders of PNB Gilts are PNB holding 74.07% and the public holds the remaining 25.93%. 127 Board of Directors The directors of PNB Gilts are Mr. S.S.Kohli, Dr. K.C. Chakrabarty, Dr. Kamal Gupta, Mr. S. K. Soni, Dr. O. P. Chawla, Mr. Mohanjit Singh, Mr. Rajendra Singh Lodha, Mr. Sunil Kant Munjal, Mr. Arun Kaul and Mr. I. D. Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital 1,350.1 1,350.1 1,350.1 Reserves and surplus 2,796.6 3,374.4 4,063.2 Total income 2,343.9 2,300.9 2,379.4 Profit after tax 1,125.9 925.1 1,069.6 Earnings per share (Rs.) 8.34 6.85 7.92 Net asset value per share (Rs.) 30.71 34.99 40.10 Promise V/s Performance PNB Gilts made a public issue in July 2000, which contained projections. A comparison of the projections as given in the prospectus with actual performance is given below. For the Year Ending March 31, 2001: (In Rs. million) Particulars Projections Actuals A. INCOME Discount on money market instruments 200.0 79.4 Interest on Government securities 1,953.0 1,741.0 Public sector units bonds 225.0 93.7 Others - 89.7 Profit on Sale of government dated securities 540.0 170.2 Sale of public sector bonds 40.0 47.5 Underwriting fee and other income 30.0 24.5 TOTAL 2,988.0 2,246.4 B. EXPENDITURES Interest on call notice & short term lending 1,546.5 1,345.6 Establishment expenses 10.4 10.7 Administrative and other expenses 28.0 23.8 Provisions against overdue call lending - 50.0 Depreciation on fixed asset 4.0 3.9 Amortisation of preliminary expenses - 5.4 TOTAL 1,588.9 1,439.5 128 (In Rs. million) Particulars Projections Actuals Profit before tax 1,399.1 806.9 Provision for tax 538.7 336.3 Profit after tax 860.4 470.5 Equity 1,350.0 1350.0 Reserves and surplus 2,499.5 2,007.9 Net worth 3,849.5 3,358.0 Earnings per share (Rs.) 6.37 3.48 Book value (Rs.) 28.52 24.87 Average net borrowings 19,000.0 14,709.5 Leverages (Average borrowings /Net owned funds) 4.94:1 4.38:1 PNB Gilts could not meet the projections as given in the prospectus for their IPO because of unfavourable market conditions in the first half of FY 2000-01, which resulted in a loss of Rs. 391.6 million. However, PNB Gilts made a profit before tax of Rs. 1198.5 million in the second half of FY 2000-01 ending the year with a profit before tax of Rs. 806.9 million. Information about Share Price The equity shares of PNB Gilts were listed on DSE, BSE and NSE. However, the company had its equity shares delisted voluntarily from DSE in March 2004 as there was no trading of its equity shares on DSE since the date of listing. PNB Gilts has complied with the clauses of the listing agreements with the Stock Exchanges. The market price of the equity shares of PNB Gilts, in the NSE during the six months preceding the filing of this Red Herring Prospectus is as follows: (In Rs.) High Low January 2005 22.60 19.50 December 2004 24.40 18.45 November 2004 18.60 16.90 October 2004 19.20 17.50 September 2004 20.75 18.75 August 2004 21.90 17.90 The market price of the equity shares of PNB Gilts as of February 7, 2005 was Rs. 20.75 at the NSE. There has been no change in the capital structure of PNB Gilts in the last six months. Investor Grievance The company attends to investor grievances/ correspondence expeditiously and usually reply is sent within 10 days of receipt, except in cases that are constrained by dispute or legal impediment. There are no investor grievances pending as of February 1, 2005. 129 PNB Asset Management Company Limited (“PNB AMC”) PNB AMC, a wholly owned subsidiary of our Bank, was incorporated on March 18, 1994. PNB AMC acted as an investment manager to PNB Mutual Fund. It was responsible for investment decisions and day-to-day operations of PNB Mutual Fund including formulations and launching of new schemes, etc. under the supervision, control and governance of the board of Trustees of the Bank’s mutual fund. We received permission from SEBI by a letter dated March 18, 2004 bearing reference number IMD/MHS/5519/2004 for the takeover and merger of the schemes of PNB Mutual Fund into pre-existing select schemes of Principal Mutual Fund and takeover of six redeemed and wound-up schemes of PNB Mutual Fund. The Board of Directors of our Bank in its meeting held on March 29, 2003 accorded in principle approval for the merger of PNB AMC with us. The memorandum of understanding between Principal Financial Services, Inc., us and Vijaya Bank on June 24, 2003 and the business transfer agreement dated August 25, 2003 between Principal AMC, Principal Trustee Company Limited, PNB AMC and the Board of trustees of PNB Mutual Fund, transferred/merged all the schemes of PNB Mutual Fund with the schemes of Principal Mutual Fund on April 30, 2004. Our application dated May 31, 2004 for deregistration of PNB Mutual Fund and de- recognition of PNB AMC is pending before SEBI. We are awaiting SEBI’s response on our application. Shareholding Pattern PNB AMC is our wholly owned subsidiary, and thus, we hold the entire share capital of PNB AMC. Board of Directors The directors of PNB AMC are Dr. K.C. Chakrabarty, Mr. D.L. Rawal, Mr. K.P. Kulkarni, Mr. S.C. Gupta and Mr. B.B. Aggarwal. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital 290.00 290.00 290.00 Reserves and surplus (-)106.70 (-)89.20 (-)77.60 Total income 41.70 35.30 40.20 Profit after tax 15.10 17.5 17.5 Earnings per share (Rs.) 0.52 0.60 0.61 Net asset value per share (Rs.) 6.32 6.92 7.53 ASSOCIATES We have nine associates, namely (1) Assets Care Enterprises Ltd. (2) UTI Asset Management Company (P) Ltd. (3) UTI Trustee Company (P) Ltd. (4) Principal PNB Asset Management Company Private Limited (5) Principal Trustee Company Private Limited (6) PNB Principal Financial Planners Private Limited (7) Principal-PNB Pensions and Life Insurance Company Limited (8) PNB- Principal Insurance Advisory Company Private Limited and (9) Everest Bank Limited. Of these, Principal PNB Insurance & Pensions Company Ltd and PNB-Principal Insurance Advisory Services Private Limited are yet to be incorporated. Assets Care Enterprises Ltd. (“Assets Care”) Assets Care was incorporated on June 11, 2002 and its registration from RBI was received on October 17, 2003. The RBI stipulated in the registration letter that the company shall commence business of securitisation/ asset reconstruction only after the RBI is satisfied that a competent person with professional experience is in place as the chief executive officer of the company. The company is in the process of appointing a chief executive officer and hence it has yet to start the business of assets reconstruction/securitisation. However, it has undertaken certain fee-based assignments, including a viability study of two companies and preparation of restructuring schemes during the financial year 2003-04. 130 Shareholding Pattern The shareholders of Assets Care, as on September 30, 2004, are Industrial Finance Corporation of India holding 33%, PNB holding 26%, Trade Finance Corporation of India holding 10.2%, Life Insurance Corporation of India holding 10%, Bank of Baroda holding 10%, United Bank of India holding 10% and Madhya Pradesh Consultancy Organisation Ltd. holding 0.8%. Board of Directors The directors of the company, as of date are Mr. Jagdish Capoor, Mr. Arvind Pande, Mr. P. B. Nimbalkar, Mr. V. V. Desai, Mr. S. C. Bhargava and Mr. R. M. Malla. Financial Performance (In Rs. million) As of March 31, 2002* 2003 2004 Capital N.A. 50.00 50 Reserves and surplus N.A. 0.60 0.01 Total income N.A. 8.09 0.36 Profit after tax N.A. 0.58 0.01 Earnings per share (Rs.) N.A. 0.12 0.002 Net asset value per share (Rs.) N.A. 10.12 10.002 * No financials since the company was incorporated in June 2002. UTI Asset Management Company (P) Ltd. (“UTI Asset Management”) UTI Asset Management was incorporated on November 14, 2002. The company is registered as an asset management company and manages the schemes of UTI Mutual Fund. Shareholding Pattern The shareholders of UTI Asset Management are SBI (through itself or nominees) holding 25%, Life Insurance Corporation of India (through itself or nominees) holding 25%, Bank of Baroda (through itself or nominees) holding 25%, PNB (through itself or nominees) holding 25%. Board of Directors The directors of the company are Mr. U.K. Sinha, Mr. S.H. Bhojani, Mr. J.S. Mathur and Mr. Vinayshil Gautam. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004* Equity share capital N.A. N.A. 100 Reserves and surplus N.A. N.A. 12,49.4 Total income N.A. N.A. 38,01.4 Profit after tax N.A. N.A. 12,49.4 Earnings per share (Rs.) NA NA 124.94 Net asset value per share (Rs.) NA NA 134.94 The first financials on the company was prepared from November 14, 2002 to March 31, 2004. 131 UTI Trustee Company (P) Ltd. (“UTI Trustee Company”) UTI Trustee Company was incorporated on November 14, 2002. The company is registered as a trustee company and are trustees of UTI Mutual Fund. Shareholding Pattern The shareholders of the company are SBI (through itself or nominees) holding 25%, Life Insurance Corporation of India (through itself or nominees) holding 25%, Bank of Baroda (through itself or nominees) holding 25%, PNB (through itself or nominees) holding 25%. Board of Directors The directors are Mr. C. Ramchandran, Dr. Kanta Ahuja, Mr. I.D. Agarwal, Mr. M. P. Radhakrishan and Dr. Pritam Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital N.A. N.A. 1 Reserves and surplus N.A. N.A. 0.029 Total income N.A. N.A. 0.057 Profit after tax N.A. N.A. 0.029 Earnings per share (Rs.) N.A. N.A. 0.29 Net asset value per share (Rs.) N.A. N.A. 10.29 The first accounting year of the company was from November 14, 2002 to March 31, 2004. Principal PNB Asset Management Company Private Limited (“Principal Asset”) Principal Asset was incorporated on November 20, 1991 as IDBI Indian Investment Company Limited. The name of the company was changed to Principal Asset Management Company Limited with effect from June 27, 2003. Subsequently, on October 20, 2003, the company was converted to a private limited company and the name of the company was changed to Principal Asset Management Company Private Limited. Further, on January 24, 2005, the name of the company was changed to Principal PNB Asset Management Company Private Limited. This company was set up to act as the asset management company for Principal Mutual Fund. The company had entered into a share sale and purchase agreement dated August 25, 2003 with Principal Financial Group (Mauritius) Limited, PNB and Vijaya Bank. The shareholders of the company have approved the proposal to change the name of the company to Principal PNB Asset Management Company Private Limited. Principal Financial Group (Mauritius) Limited has decided to dilute its stake in the company from 65% to 60% and offer 5% stake to Andhra Bank. Accordingly an MOU has been signed amongst the current shareholders and Andhra Bank. Shareholding Pattern The shareholders of Principal Asset are PNB holding 30%, Vijaya Bank holding 5% and Principal Financial Group (Mauritius) Limited holding 65%. Board of Directors The directors of the company are Mr. Arpan Thanawala, Mr. Sanjay Sachdev, Mr. Rajat Jain, Mr. Rustam Gagrat, Dr. Amit Mitra and Mr. Arun Kaul. 132 Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital 100.00 100.00 100.00 Reserves and surplus 502.76 557.40 552.97 Total income 174.28 223.71 233.80 Profit after tax 1.46 57.66 51.97 Earnings per share (Rs.) 0.15 5.77 5.19 Net asset value per share (Rs.) 60.28 65.74 65.30 Principal Trustee Company Private Limited (“Principal Trustee Company”) Principal Trustee Company was incorporated on November 6, 2000 as IDBI-Principal Trustee Company Limited and the name was changed to Principal Trustee Company Limited with effect from June 27, 2003. Subsequently on October 20, 2003, the company was converted into a private limited company and its name was changed to Principal Trustee Company Private Limited. It was established to act as the trustee company for the Principal Mutual Fund. Shareholding Pattern The shareholders of Principal Trustee Company are PNB holding 30%, Principal Financial Group (Mauritius) Limited holding 65% and Vijaya Bank holding 5%. Board of Directors The directors of the company are Mr. B.G. Deshmukh, Dr. S. A. Dave, Mr. S. Ravi, Mr. D. L. Rawal, Mr. H.M.Singh and Mr. Norman Sorensen. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Equity share capital 1.00 1.00 1.00 Reserves and surplus -0.08 0.38 1.99 Total income 0.00 0.89 2.75 Profit after tax -0.08 0.47 1.61 Earnings per share (Rs.) 0.8 4.7 16.1 Net asset value (Rs.) 9.2 13.8 29.9 * The first financial statements were made for the period November 6, 2000 to December 31, 2001. The subsequent financial statements were prepared for the period January 1, 2002 to March 31, 2003. PNB Principal Financial Planners Private Limited (“PNB Principal Financial”) PNB Principal Financial has been incorporated on October 11, 2004. We have entered into a share subscription agreement with Principal Financial Group Mauritius Ltd. and Vijaya Bank on August 25, 2003 for setting up this joint venture company. The objectives of the company as stated in the agreement to establish a company for the distribution of financial products. We have also entered into a shareholders agreement with Principal Financial Group Mauritius Ltd. and Vijaya Bank on October 14, 2004 which lays down the respective rights and duties of the shareholders of the company. Shareholding Pattern The shareholders of PNB Principal Financial are Principal Financial Group Mauritius Limited holding 65%, PNB holding 30% and Vijaya Bank holding 5%. 133 Board of Directors The directors of PNB Principal Financial are Mr. Sudhin Padhye, Mr. Ritesh Jain, Mr. Subhashish Sharma, Mr. R.I.S. Sidhu, Mr. P.K. Mitra and Mr. Ratnakar Hegde. Principal-PNB Pensions and Life Insurance Company Limited We have entered into a memorandum of understanding dated June 24, 2003 with Principal Financial Group of USA for setting up the above mentioned joint venture in partnership with Vijaya Bank and Berger Paints Limited. The company is yet to be incorporated. However we have informed RBI vide letter dated October 18, 2004 that the proposed name of the company is Principal-PNB Pensions and Life Insurance Company Limited. Shareholding Pattern The shareholders of Principal-PNB Pensions and Life Insurance Company Limited are PNB holding 37%, Principal Financial Group holding 26%, Vijaya Bank holding 12% and Berger Paints Limited holding 25%. PNB Principal Insurance Advisory Company Private Limited We have entered into a memorandum of understanding dated June 24, 2003 with Principal Financial Group of USA with Vijaya Bank and Berger Paints Limited for setting up the joint venture. Shareholding Pattern The shareholders of PNB Principal Insurance Advisory Company Private Limited are PNB holding 30%, Principal Financial Group holding 26%, Vijaya Bank holding 19% and Berger Paints Limited holding 25%. The company is yet to be incorporated. We have referred to the name of the company as ‘PNB Principal Insurance Advisory Company Private Limited’ in letter dated October 19, 2004 addressed to the RBI. Everest Bank Limited (“Everest Bank”) Everest Bank was incorporated in Kathmandu under the laws of Nepal on October 18, 1994. We entered into a Technical Services (Equity & Management Participation) Agreement with the Everest Bank. Under the terms of this agreement we provide organisational set up, systems and procedures of operation and control and supervision and development of personnel with a view to ensuring a sound, efficient and internationally accepted standard of service. Shareholding Pattern The shareholders of Everest Bank are Mr. B. K. Shrestha, Mr. M. D. Pathak, Mr. Bhagwanlal Shrestha, Mr. N. K. Shrestha, Mr. Arun Man Sherchan, Mr. K. B. Tuladar and Hotel Snowlion who are the Nepalese promoters to the extent 50%, the general public to the extent of 30% and PNB to the extent of 20%. Board of Directors The directors are Mr. B. K. Shrestha, Mr. Arun Man Sherchan, Mr. Bal Gopal Baidya, Mr. Ranjan Dhawan, Mr. Nabin Bhakta Shrestha, Mr. Shiva Sharan K. C., Mr. V. K. Shrestha and Mr. D.V.S.S.Prasad. Financial Performance (In Nepali Rupiya in million)* As on July, 2002 2003 2004 Equity share capital 399.32 455.0 455.0 Reserves and surplus 131.59 157.82 225.32 Total income 540.93 635.33 785.06 Profit after tax 85.35 94.18 143.57 Earnings per share 21.37 20.70 31.55 Net asset value per share 132.95 134.69 149.52 * As on February 11, 2005, the buying price for Rs. 100 is quoted at 160.0 Nepali Rupaiya as per exchange rates fixed by the Nepal Rastra Bank . 134 Promise V/s Performance (In Nepali Rupiya million)* Projected on last Public Offer Audited Actual Figures Growth Percentage (%) 2001/ 2002/ 2003/ 2001/ 2002/ 2003/ 2001/ 2002/ 2003/ 2002 2003 2004 2002 2003 2004 2002 2003 2004 Income Income 419.42 457.17 502.89 443.821 520.173 657.249 5.82 13.78 30.69 Commission & Discount 31.080 35.742 41.103 36.773 61.504 74.331 18.32 72.08 80.84 Other Income 57.430 66.044 75.950 60.337 53.655 53.479 5.06 -18.76 -29.59 Total Income 507.930 558.956 619.943 540.931 635.332 785.059 6.50 13.66 26.63 Expense Expenses 260.377 286.415 315.057 257.051 307.639 316.366 -1.28 7.41 0.42 Staff Expenses 28.600 31.460 34.606 32.187 37.368 48.530 12.54 18.78 40.24 Operating Expenses 55.500 61.050 67.155 75.426 93.585 103.807 35.90 53.29 54.58 Net Operating Expenses 4.000 4.200 4.500 -100.00 -100.00 -100.00 Total Expenses 348.477 383.125 421.318 364.664 438.592 468.703 4.65 14.48 11.25 Operating Profit 159.453 175.831 198.625 176.267 196.740 316.356 10.54 11.89 59.27 Less Provisions & exp 57.885 68.523 81.243 48.883 60.845 105.237 -15.55 -11.20 29.53 Profits Before Tax 101.568 107.308 117.382 127.384 135.895 211.119 25.42 26.64 79.86 Provision for Tax 33.517 35.412 38.736 42.037 41.714 67.551 25.42 17.80 74.39 68.051 71.896 78.646 85.347 94.181 143.568 25.42 31.00 82.55 Preference Share Dividend 12.600 12.600 12.600 12.600 0.00 0.00 Net Profit after Preference 68.051 59.296 66.046 85.347 81.581 130.968 25.42 37.58 98.30 As on February 11, 2005, the buying price for Rs. 100 is quoted at 160.0 Nepali Rupaiya as per exchange rates fixed by the Nepal Rastra Bank. Information about Share Price The equity shares of Everest Bank are listed on the Nepal Stock Exchange. Everest Bank has complied with the clauses of the listing agreement with the Nepal Stock Exchange. 135 The market price of the shares during the preceding six months at Nepal Stock Exchange is as described in the table below: (In Nepali Rupaiya) Period High Low December 2004 700 650 November 2004 685 625 October 2004 700 680 September 2004 701 665 August 2004 739 671 July 2004 710 660 June 2004 643 552 May 2004 551 516 April 2004 545 488 * As on February 11, 2005, the buying price for Rs. 100 is quoted at 160.0 Nepali Rupaiya as per exchange rates fixed by the Nepal Rastra Bank. The high and low market price of the equity shares of Everest Bank as on February 11, 2005 is as follows: (In Nepali Rupaiya)* High Low 702 688 * As on February 11, 2005, the buying price for Rs. 100 is quoted at 160.0 Nepali Rupaiya as per exchange rates fixed by the Nepal Rastra Bank. The closing share price of equity shares of Everest Bank as on January 7, 2005 was 652 Nepali Rupaiya. There has been no change in the capital structure of Everest Bank in the last six months. Investor Grievance Everest Bank does not have any pending grievance of investors as of December 31, 2004. 136 REGIONAL RURAL BANKS (“RRBS”) The Bank has sponsored 19 RRBs with a network of 1,288 branches spread over 55 districts in seven states of India i.e. Bihar, Haryana, Himachal Pradesh, Punjab, Rajasthan, Uttranchal and Uttar Pradesh. The equity share capital of each of the RRBs is held to the extent of 50% by the Government, 15% by the respective State government and 35% by the Bank. The names of the RRBs are as follows: (1) Bhojpur-Rohtas Gramin Bank; (2) Magadh Gramin Bank; (3) Nalanda Gramin Bank; (4) Patliputra Gramin Bank; (5) Haryana Kshetriya Gramin Bank; (6) Hisar-Sirsa Kshetriya Gramin Bank; (7) Ambala-Kurukshetra Gramin Bank; (8) Himachal Gramin Bank; (9) Shivalik Kshetriya Gramin Bank; (10) Kapurthala-Firozpur Kshetriya Gramin Bank; (11) Gurdaspur-Amritsar Kshetriya Gramin Vikas Bank; (12) Shekhawati Gramin Bank; (13) Alwar-Bharatpur Anchalik Gramin Bank; (14) Kisan Gramin Bank; (15) Devi Patan Kshetriya Gramin Bank; (16) Rani Lakshmibai Kshetriya Gramin Bank; (17) Vidur Gramin Bank (18) Muzaffarnagar Kshetriya Gramin Bank; and (19) Hindon Gramin Bank. As of December 31, 2004, our Bank has deputed 56 officers to RRBs. Our Bank is running a training centre at Lucknow, India to train the staff of these RRBs. Bhojpur-Rohtas Gramin Bank The Bhojpur-Rohtas Gramin Bank was incorporated on December 26, 1975. Board of Directors The directors of Bhojpur-Rohtas Gramin Bank are Mr.C.K. Jha, Mr. C.B. Awasthi, Mr. B. B. Malhotra, Mr. Gulab Chand, Mr. Mohammed Ehtesham, Mr. R.Paswan, Mr. Kameshwar Prasad Singh, Mr. Baban Singh Yadav and Mr. Prabhat Kumar. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 231.30 231.30 231.30 Reserves and surplus 132.76 288.18 453.86 Deposits 6177.06 6817.01 7583.84 Advances 1311.79 1636.66 2071.60 Total income 822.32 771.00 756.67 Total expenditure 705.05 618.51 590.99 Profit after tax 117.28 152.48 165.68 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. 1172.8 1524.8 1656.8 NAV per share in Rs. 3740.6 5294.8 6951.6 Magadh Gramin Bank The Magadh Gramin Bank was incorporated on November 10, 1976. 137 Board of Directors The directors of Magadh Gramin Bank are Mr. Ramayan Tiwari, Mr. K. I. Singh, Mr. B. L. Sebastian, Mr. Mahadeo Prasad, Mr. Vinay Kumar Sharma, Mr. B. K. Thakur, Mr. Raj Bahadur Singh, Dr. C. Bhashkararan and the deputy development commissioner of Gaya. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 255.00 255.00 255.00 Reserves and surplus 202.37 368.13 548.68 Deposits 5338.07 5508.63 5872.94 Advances 837.54 969.40 1079.98 Total income 703.66 718.05 681.84 Total expenditure 515.18 551.46 499.30 Profit after tax 188.48 166.59 182.84 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.03 0.00 EPS in Rs. 1884.8 1665.9 1828.4 NAV per share in Rs. 4673.7 6331.3 8136.8 Nalanda Gramin Bank The Nalanda Gramin Bank was incorporated on March 31, 1979. Board of Directors The directors of Nalanda Gramin Bank are Mr. D. Singh, Mr. C. B. Awasthi, Mr. N.S.Jain, Mr. A.K.Verma, Mr. Innocent Soren, Mr. Gulab Chand, Mr. Tarkeshwar Rai, Mr. Md. Arshad and Mr. Bijendra Deo. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 73.02 73.02 73.02 Reserves and surplus 0.00 1.29 0.00 Deposits 2321.26 2606.26 2540.09 Advances 403.52 441.54 531.11 Total income 192.08 227.28 211.18 Total expenditure 199.92 211.60 205.97 Profit after tax -7.84 15.67 5.21 Accumulated losses 443.23 432.82 432.42 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. -78.4 156.7 52.1 NAV per share in Rs. 830.2 843.1 830.2 138 Patliputra Gramin Bank The Patliputra Gramin Bank was incorporated on November 27, 1984. Board of Directors The directors of Patliputra Gramin Bank are Mr. J.B.Singh, Mr. B. C. Nigam, Mr. Kumar Rahul, Mr. B. K. Thakur, Mr. Arun Kumar Sinha, Mr. R. B. Singh, Ms. Esabela Bage, Mr. B. Raghunath and Mr. K. B. Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 33.91 33.91 33.91 Reserves and surplus 3.69 22.72 48.07 Deposits 660.41 680.13 848.01 Advances 182.84 243.66 315.29 Total income 69.78 76.35 82.61 Total expenditure 51.05 57.32 57.25 Profit after tax 18.73 19.03 25.35 Accumulated losses 15.04 0.00 0.00 Contingent liabilities 0.68 0.68 0.68 EPS in Rs. 187.3 190.3 253.5 NAV per share in Rs. 476.0 666.3 919.8 Haryana Kshetriya Gramin Bank The Haryana Kshetriya Gramin Bank was incorporated on October 2, 1975. Board of Directors The directors of Haryana Kshetriya Gramin Bank consists of Mr. Vivek Arya, Mr. A.K. Verma, Mr. P.C. Mohanpuria, Mr. N.K. Verma, Mr. Pawan Kumar, Mr. Rishi Prakash Sharma, Mr. Suraj Mal, Mr. Kuljas Rai and Mr. Surjit Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 140.09 140.09 140.09 Reserves and surplus 6.35 4.81 0.00 Deposits 3283.16 3639.51 4214.90 Advances 1585.19 1940.80 2520.74 Total income 354.50 421.29 457.80 Total expenditure 317.96 342.13 327.43 Profit after tax 36.54 79.17 130.37 Accumulated losses 250.05 170.88 40.51 Contingent liabilities 5.20 4.14 0.00 EPS in Rs. 365.4 791.7 1303.7 NAV per share in Rs. 1564.4 1549 1500.9 139 Hissar Sirsa Kshetriya Gramin Bank The Hissar Sirsa Kshetriya Gramin Bank was incorporated on October 2, 1984. Board of Directors The directors of Hissar Sirsa Kshetriya Gramin Bank are Mr. S. P. Kachhal, Mr. K. C. Salota, Mr. O. P. Goyal, Mr. Sunil Kumar, Mr. Pawan Kumar, Dr. R. K. Patel, Mr. Giriraj Dagar, Mr. T. Ranganthan and Mr. Pankaj Aggarwal. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 41.89 41.89 41.89 Reserves and surplus 107.99 151.14 206.92 Deposits 1200.63 1385.86 1608.90 Advances 924.26 1080.00 1293.01 Total income 174.87 191.70 221.30 Total expenditure 142.52 148.55 165.52 Profit after tax 32.35 43.15 55.78 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 6.10 6.10 6.23 EPS in Rs. 323.5 431.5 557.8 NAV per share in Rs. 1598.8 2030.3 2588.1 Ambala Kurukshetra Gramin Bank Ambala Kurukshetra Gramin Bank was incorporated on January 18, 1985 Board of Directors The directors of Ambala Kurukshetra Gramin Bank are Mr. Jagir Singh, Mr. H. L. Arora, Mr. R. S. Sharma, Mrs. Sudesh Vatts, Mr. R. K. Singh, Ms. Keshni Anand Arora, Mr. M. L. Kaushik, Mr. Pawan Kumar and Mr. Ram Prasad. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 0.00 49.09 49.09 Reserves and surplus 22.76 43.18 82.14 Deposits 1210.18 1338.89 1447.20 Advances 764.57 885.75 1017.17 Total income 148.21 151.44 156.15 Total expenditure 130.07 131.02 117.19 Profit after tax 18.14 20.42 38.96 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. 181.4 204.2 389.6 NAV per share in Rs. 327.6 1022.7 1412.3 140 Himachal Gramin Bank The Himachal Gramin Bank was incorporated on December 23, 1976. Board of Directors The directors of Himachal Gramin Bank are Mr. Gurdial Singh, Mr. R. P. Sharma, Mr. R. S. Sharma, Mr. J. C. Sharma, Mr. R. S. Gupta, Mr. Nirmal Singh Suman, Dr. Onkar Shad, Mr. B. K. Sharma and Mr. Malkiat Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 104.00 104.00 106.53 Reserves and surplus 88.55 151.17 264.01 Deposits 4924.04 5464.81 5788.27 Advances 1312.43 1615.63 1373.89 Total income 587.25 550.33 569.14 Total expenditure 521.45 521.50 481.28 Profit after tax 65.80 28.83 87.86 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.50 0.15 EPS in Rs. 658.0 288.3 878.6 NAV per share in Rs. 2025.5 2651.7 3805.4 Shivalik Kshetriya Gramin Bank The Shivalik Kshetriya Gramin Bank was incorporated on March 30, 1983. Board of Directors The directors of Shivalik Kshetriya Gramin Bank are Dr. Phool Singh, Mr.Ved Vyas, Mr. R. S. Virmani, Mr. Jaipal, Mr. Raghubir Singh, Mrs. Amarjit Kaur, Mr. D. R. Bhagat, Mr. J. R. Mahajan and Mr. C. K. Bajaj. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 15.81 15.81 15.81 Reserves and surplus 254.97 345.50 458.03 Deposits 1925.22 2176.12 2454.77 Advances 497.33 629.14 801.10 Total income 256.38 270.74 297.61 Total expenditure 181.28 180.21 198.58 Profit after tax 75.10 90.52 99.03 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.08 0.48 0.45 EPS in Rs. 751.0 905.2 990.3 NAV per share in Rs. 2807.8 3713.1 4838.4 141 Kapurthala-Firozpur Kshetriya Gramin Bank Kapurthala-Firozpur Kshetriya Gramin Bank was incorporated on March 30, 1983. Board of Directors The directors of Kapurthala-Firozpur Kshetriya Gramin Bank are Mr. R. C. Yadav, Mr. R. R. P. Sharda, Mr. Harnek Singh, Mr. Amrit Lal Chhabra, Mr. Chand Singh, Mrs. Amarjit Kaur, Mr. N. N. Sharma, Mrs. Shashi Mahajan and Mr. Ravindra Kumar. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 92.46 92.46 92.46 Reserves and surplus 10.01 95.82 168.94 Deposits 1215.56 1501.84 1855.14 Advances 438.83 568.12 708.76 Total income 177.19 218.71 214.48 Total expenditure 126.72 132.89 141.37 Profit after tax 50.47 85.81 73.12 Accumulated losses 40.47 0.00 0.00 Contingent liabilities 0.17 1.06 0.05 EPS in Rs. 504.7 858.1 731.2 NAV per share in Rs. 1124.7 1982.8 2714 Gurdaspur Amritsar Kshetriya Gramin Vikas Bank The Gurdaspur Amritsar Kshetriya Gramin Vikas Bank was incorporated on March 31, 1983. Board of Directors The directors of Gurdaspur-Amritsar Kshetriya Gramin Vikas Bank are Mr. R.S.Kalaria, Mr. Jagat Ram, Mr. S. K. Mohla, Mr. Sohan Singh, Mr. M. R. Garg, Mrs. Amarjit Kaur, Mr. M.P Singh, Dr. Dilbagh Rai and Mr. Keshav Nand Sharma. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 86.72 86.72 86.72 Reserves and surplus 206.61 326.95 467.61 Deposits 2510.25 2850.73 2884.73 Advances 839.76 1097.95 1311.52 Total income 337.90 358.84 363.49 Total expenditure 230.23 238.49 222.83 Profit after tax 107.67 120.34 140.66 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 1.81 1.08 1.08 EPS in Rs. 1076.7 1203.4 1406.6 NAV per share in Rs. 3033.3 4236.7 5643.3 142 Shekhawati Gramin Bank The Shekhawati Gramin Bank was incorporated on October 7, 1976. Board of Directors The directors of Shekhawati Gramin Bank are Mr. S.K. Singhvi, Mr. V. K. Gupta, Mr. R. S. Verma, Mr. S. K. Lohra, Mr. B. L. Meena, Mr. Mahesh Bhardwaj, Mr. V. K. Runthala, Mr. Suresh Jain and Mr. Bhiwanram Chaudhary. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 295.29 306.28 306.28 Reserves and surplus 7.20 7.20 116.48 Deposits 3585.93 3777.78 4146.80 Advances 1073.70 1217.34 1495.91 Total income 449.68 487.21 531.35 Total expenditure 360.63 372.50 399.65 Profit after tax 89.05 114.71 131.70 Accumulated losses 129.93 15.22 0.00 Contingent liabilities 1.82 3.44 4.00 EPS in Rs. 890.5 1147.1 1317.0 NAV per share in Rs. 3124.9 3234.8 4327.6 Alwar-Bharatpur Anchalik Gramin Bank Alwar-Bharatpur Anchalik Gramin Bank was incorporated on February 28, 1981. Board of Directors The directors of Alwar-Bharatpur Anchalik Gramin Bank are Mr. C. B. Bhasin, Mr. S. K. Lohra, Mr. Satpal Bhagtiar, Mr. S. L. Gurjar, Mr. B. S. Khatri, Mr. V. K. Gupta, Mr. C.M.Kala and Mr. R. H. S. Yadav. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 113.73 113.73 133.80 Reserves and surplus 0.00 0.00 50.29 Deposits 2416.42 2800.15 3295.74 Advances 1285.86 1581.38 2004.83 Total income 329.31 370.96 404.29 Total expenditure 259.05 273.81 266.04 Profit after tax 70.26 97.15 138.25 Accumulated losses 185.11 87.96 0.00 Contingent liabilities 0.97 1.29 1.01 EPS in Rs. 702.6 971.5 1382.5 NAV per share in Rs. 1237.3 1237.3 1940.9 143 Kisan Gramin Bank The Kisan Gramin Bank was incorporated on May 19, 1980. Board of Directors The directors of Kisan Gramin Bank are Mr. N.K. Kacker, Mr. V. P. Jain, Mr. S.C. Arora, Mr. S.P. Yadav, Mr. Arun Bhagoliwal Mr. R. K. Yadav, Mr. A. K. Chauhan, Mr. Avnesh Kumar and Mr. Narendra Pal Singh. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 155.39 155.39 163.97 Reserves and surplus 0.00 0.00 0.00 Deposits 1200.94 1279.62 1378.14 Advances 494.70 597.09 683.35 Total income 157.42 160.62 168.30 Total expenditure 125.68 134.19 136.72 Profit after tax 31.74 26.42 31.58 Accumulated losses 101.92 75.50 43.92 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. 317.4 264.2 315.8 NAV per share in Rs. 1653.9 1653.9 1739.7 Devi Patan Kshetriya Gramin Bank The Devi Patan Kshetriya Gramin Bank was incorporated on January 17, 1981. Board of Directors The directors of Devi Patan Kshetriya Gramin Bank are Mr. P.R.Misra, Mr. U. K. Gupta, Mr. S. K. Gakhar, Mr. Bhav Nath Singh, Mr. Ramakant Tewari, Mr. Ajay Misra, Mr. A. K. Srivastava, Mr. Pramod Kumar and Mr. Babu Lal Bharti. 144 Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 139.75 139.75 139.75 Reserves and surplus 153.21 223.82 315.50 Deposits 3025.46 3499.15 4021.89 Advances 771.04 928.57 902.37 Total income 318.73 328.09 352.95 Total expenditure 243.28 257.47 261.27 Profit after tax 75.46 70.61 91.68 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.10 1.02 1.11 EPS in Rs. 754.6 706.1 916.8 NAV per share in Rs. 3029.6 3735.7 4652.5 Rani Laxmibai Kshetriya Gramin Bank Rani Laxmibai Kshetriya Gramin Bank was incorporated on March 31, 1982. Board of Directors The directors of Rani Laxmibai Kshetriya Gramin Bank are Mr. Girwar Singh, Mr. V.P. Garg, Mr. Arun Bhagoliwal Mr. J. P. Gupta, Mr. S. K. Srivastava, Mr. M. A. Kidwai, Mr. Sunil Kumar Yadav, Mr. Jaswant Singh Solanki and Mr. Rajesh Sahu. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 0.00 0.00 0.00 Reserves and surplus 0.00 0.00 0.00 Deposits 1002.27 1144.89 1308.17 Advances 367.07 470.13 564.01 Total income 81.83 109.82 200.16 Total expenditure 109.21 108.26 197.19 Profit after tax -27.39 1.56 2.98 Accumulated losses 358.27 356.71 353.73 Contingent liabilities 1.01 0.98 0.96 EPS in Rs. -273.9 15.6 29.8 NAV per share in Rs. 100.0 100 100 145 Vidur Gramin Bank Vidur Gramin Bank was incorporated on January 18, 1983. Board of Directors The directors of Vidur Gramin Bank are Mr. A. K. Gupta, Mr. Ramesh Kumar, Mr. Rajendra Mahajan, Mr. Ashok Verma, Mrs. Anita Singh, Mr. Rajiv Kumar Agrawal, Mr. K. S. Bhatti, Mr. V. K. Goel and Mr. Kalika Prasad. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 79.26 79.26 80.95 Reserves and surplus 55.32 105.53 155.71 Deposits 1304.59 1402.62 1667.18 Advances 436.55 529.09 608.02 Total income 161.96 168.21 179.31 Total expenditure 111.79 118.00 129.13 Profit after tax 50.17 50.21 50.18 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 2.71 12.89 6.72 EPS in Rs. 501.7 502.1 501.8 NAV per share in Rs. 1445.8 1947.9 2466.6 Muzaffarnagar Kshetriya Gramin Bank Muzaffarnagar Kshetriya Gramin Bank was incorporated on July 27, 1984. Board of Directors The directors of Muzaffarnagar Kshetriya Gramin Bank are Mr. B.B.Vohra, Mr. A. K. Patnayak, Mr. D. K. Jain, Mr. Sushil Kumar Gakhar, Mr. R. S. Lal, Mr. Vijay Pal Singh, Mr. Rajveer Singh and Mr. Ramphal Singh. 146 Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 39.17 39.17 41.19 Reserves and surplus 31.38 57.89 80.10 Deposits 747.12 779.14 903.09 Advances 281.72 329.50 378.09 Total income 97.85 97.51 92.32 Total expenditure 66.74 71.00 70.10 Profit after tax 31.11 26.51 22.22 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. 311.1 265.1 222.2 NAV per share in Rs. 805.5 1070.6 1311.9 Hindon Gramin Bank Hindon Gramin Bank was incorporated on March 28, 1987. Board of Directors The directors of Hindon Gramin Bank are Mr. S.M.Shastri, Mr. S. K. Sinha, Mr. S. S. Yadav, Mr. Deepak Agarwal, Mr. Om Bir Singh, Mr. Pradeep Garg, Mr. V.J. Matto, Mr. V. K. Bajpayee and Dr. Shishir Sharma. Financial Performance (In Rs. million) As of March 31, 2002 2003 2004 Capital 10.00 10.00 10.00 Share capital deposit account 31.38 31.38 32.02 Reserves and surplus 17.92 33.43 52.20 Deposits 527.63 577.16 658.47 Advances 203.80 267.89 320.75 Total income 64.09 66.36 68.19 Total expenditure 48.55 49.83 49.43 Profit after tax 15.55 16.53 18.77 Accumulated losses 0.00 0.00 0.00 Contingent liabilities 0.00 0.00 0.00 EPS in Rs. 155.5 165.3 187.7 NAV per share in Rs. 593.0 748.10 942.2 147 INFORMATION ON THE GROUP COMPANIES WHICH HAVE BEEN CLOSED IN THE LAST FIVE YEARS. Two of our group companies have been closed in the last five years. The details of the same are as follows: PNB Capital Services Limited PNB Capital Services Limited was a wholly owned subsidiary of our Bank and offered a wide range of services including merchant banking, syndication of loans, commercial papers and external commercial borrowings, debenture trusteeship, advisory services and project appraisal. It also offered services such as placement of debt, advice on mergers, acquisitions and takeovers. RBI had advised closure of PNB Capital Services Limited by way of its letter dated November 8, 1999, in view of the lack of clarity on sustainability of non fund based business. The Bank requested RBI at various points in time to reconsider the matter and accord approval for the company to continue and enter into a joint venture with a strategic partner. The RBI however declined this request and asked the Bank to close down PNB Capital Services Limited at the earliest. In terms of the scheme of amalgamation, approved by the Board of the Bank in its meeting held on March 1, 2003 and sanctioned by the High Court of Delhi vide order dated August 25, 2003, appropriate filings were made with the Registrar of Companies and PNB Capital Services Limited was merged with the Bank with effect from April 1, 2003. PNB Capital Services Limited was associated with the public issue of shares of Maha Chemicals Ltd. in April, 1994 as merchant banker. Maha Chemicals Ltd. appears in the list of vanishing companies as identified by SEBI. SEBI had issued a show cause notice in this respect to PNB Capital Services Limited, and post their reply to the show cause notice had directed PNB Capital Services Ltd. vide its letter dated September 7, 2001 to be more careful in future. As stated above, PNB Capital Services Limited has merged with us and is no longer in existence as a separate entity. PNB Securities Ltd. PNB Securities Ltd. was incorporated on June 13, 1996 as a wholly owned subsidiary of the Bank with the objective of undertaking the brokerage business. The subscribed capital of the company was Rs. 5,000,000. However, the company, since its inception transacted no business and the company was continuing as a defunct company. The company passed a special resolution for voluntary winding up u/s 484(1)(b) of the Companies Act on August 16, 2001. A liquidator has been appointed and income tax dues have been paid up to March 31, 2001. The assets and liabilities of the company were liquidated in accordance with the liquidator’s statement of accounts dated February 25, 2002 in Form 156 filed with the appropriate Registrar of Companies. The capital of the Bank was returned on March 9, 2002. The Office of the Official Liquidator, Ministry of Law, Justice and Company Affairs, vide letter dated December 24, 2002 has referred the matter to the registrar of companies for the dissolution of the company. The response of the registrar of companies is awaited for final winding up orders. 148 RELATED PARTY TRANSACTIONS We have entered into related business transactions with key managerial personnel and associates. As per the RBI circular no. DBOD.No.BP. BC. 89 /21.04.018/2002-03 dated March 29, 2003 on Guidelines on the Compliance of Accounting Standard by Banks, all nationalized banks are exempt from disclosing their transactions with their subsidiaries as well as the RRBs sponsored by them. Accordingly we are disclosing transactions with key management personnel in the audited accounts in compliance with the relevant accounting standard and extant RBI circular. Transactions with whole time directors (Mr. S. S. Kohli, Chairman and Managing Director and Mr. T. S. Narayanaswami, erstwhile Executive Director), who have been termed key managerial personnel for the purposes of the audited accounts of the Bank, are as follows (These numbers are from the audited accounts): In Rs. million Item Amount Amount Amount (2001-02) (2002-03) (2003-04) Remuneration 0.9 1.0 1.1 Transactions with our associates are as follows (These numbers are unaudited): UTI Asset Management Company (P) Ltd. There have been no transactions between the Bank and this associate. UTI Trustee Company (P) Ltd. There have been no transactions between the Bank and this associate. Principal-PNB Pensions and Life Insurance Company Limited There have been no transactions between Bank and this associate. PNB-Principal Insurance Advisory Company Private Limited There have been no transactions between the Bank and this associate. Assets Care Enterprises Ltd. Rs. in million FY 2002 FY 2003 FY 2004 Share Capital held by Bank - - 13 Principal PNB Asset Management Company Private Limited Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Share Capital held by Bank - - 30 149 Principal Trustee Company Private Limited Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Share Capital held by Bank - - 0.30 PNB Principal Financial Planners Private Limited A sum of Rs. 15,000,000 was paid to this company on October 14, 2004 by our Head Office, Treasury Division, towards the Bank’s share of capital contribution. Further, the company has paid a sum of Rs. 0.9 million royalty for use of the Bank’s name. Rs. in million FY 2003 FY 2004 FY 2005 (as of date) Capital Contribution to joint venture - - 15 Royalty to the Bank - - 0.9 Everest Bank Limited Rs. in million FY 2002 FY 2003 FY 2004 Share Capital held by Bank 33.0 39.6 39.6 Dividend received by Bank - 7.9 7.9 Technical Services fee received by Bank 2 2 2 150 REGULATIONS AND POLICIES The main legislation governing commercial banks, the Banking Regulation Act, applies to public sector banks like Punjab National Bank only to a limited extent. Sections 34A, 36AD and Section 51 of the Banking Regulation Act are applicable to corresponding new banks constituted under the Bank Acquisition Act. In turn, Section 51 of the Bank Acquisition Act makes some of its sections applicable to corresponding new banks. Punjab National Bank, as a corresponding new bank, is governed primarily by the provisions of the Bank Acquisition Act. The Bank Scheme and the Bank Regulations govern our operations. Other important laws include the Reserve Bank of India Act, 1934. Additionally, the RBI, from time to time, issues guidelines, regulations, policies, notifications, press releases, circulars, etc. to be followed by us and supervises our compliance with these guidelines. Like all corresponding new banks, we are regulated and supervised by the RBI. The RBI requires us to furnish statements, information and certain details relating to our business. It has issued guidelines for on several matters including recognition of income, classification of assets, valuation of investments, maintenance of capital adequacy and provisioning for impaired assets. The RBI has set up a Board for Financial Supervision (“BFS”), under the chairmanship of the Governor of the RBI. The primary objective of the BFS is to undertake consolidated supervision of the financial sector comprising commercial banks, financial institutions and non banking finance companies. The appointment of the auditors of banks is subject to the approval of the RBI. The Companies Act does not apply to us and therefore there are important differences in the rights that are available to a shareholder under the Companies Act and the rights available to a shareholder of a corresponding new bank under the provisions of our Constitutional Documents. The table provided below summarises these differences. COMPARATIVE TABLE OF RIGHTS OF SHAREHOLDERS UNDER COMPANIES ACT, 1956 AND UNDER REGULATIONS APPLICABLE TO CORRESPONDING NEW BANKS* COMPANY CORRESPONDING NEW BANK Co. Act Rights Provision of Constitutional Important Rights Documents 38 Not to be bound by an alteration made in No corresponding the memorandum of association/articles of provision. association after the date of becoming a member, so far as the alteration requires him to subscribe for more shares, or increases his liability to contribute to the share capital, or otherwise to pay money to the company. 39 To require a company to send within seven No corresponding days of the requisition, a copy of each of provision. the following documents as in force for the time being: (a) the Memorandum of Association; (b) the Articles of Association; arid (c) every agreement and every resolution referred to in Section 192, if in so far as they have not been embodied in the memorandum or articles. 151 49 To inspect the register of investments No corresponding provision. and to petition the Central Government if the inspection is refused. 53 To be served with a document by the Regulation 46 Regulation 46: Service of a notice or company. document to shareholders. The Bank may serve a notice or a document on any shareholder either personally, or by ordinary post at his registered address or if he has no registered address in India, at the address, if any, within India as supplied by the shareholder. 62 To sue directors, promoters or persons No corresponding provision. who have authorized the issue of the prospectus for loss or damage suffered by reason of any untrue statement included in the prospectus. 71 To avoid irregular allotment of shares/ No corresponding provision. debentures. 73 To obtain repayment of the application No corresponding provision. money/excess application money. 81 Rights in relation to rights issue and No corresponding provision. preferential allotment. 84 To receive a share certificate and obtain a Regulations 14 and 15 Issue of Share Certificates. Issue procedure duplicate if the original lost or damaged. of share certificate and joint share certificates, duplicate share certificates detailed. 87 Voting rights at general meetings and on Regulations 61 and 68 Regulation 61: Voting at general meetings a poll in proportion to the share of the Sections 3(2BBA)(a) and (i) At any general meeting, a resolution put paid-up equity capital of the company. 3(2E) to the vote of the meeting shall, unless a poll is demanded, be decided on a show of hands. (ii) Save as otherwise provided in the Act every matter submitted to a general meeting shall be decided by a majority of votes. (iii) Before or on the declaration of the result of the voting on any resolution on a show of hands, a poll may be ordered to be taken by the chairman of the meeting of his own motion and shall be ordered to be taken by him on a demand made in that behalf by any shareholder or shareholders present in person or by proxy and holding shares in 152 the bank which confer a power to vote on the resolution not being less than one fifth of the total voting power in respect of the resolution. Regulation 68: Determination of voting rights (i) Subject to the provisions contained in Section 3 (2E) of the Act, each shareholder shall, at such meeting, have one vote on show of hands and in case of a poll shall have one vote for each share held by him. (ii) Subject to the provisions contained in Section 3 (2E) of the Act, every shareholder entitled to vote as aforesaid who, not being a company, is present in person or by proxy or who being a company is present by a duly authorised representative, or by proxy shall have one vote on a show of hands and in case of a poll shall have one vote for each share held by him as stated hereinabove in sub-regulation (i) Explanation - For this Chapter, “Company” means any body corporate. (iii) Shareholders of the bank entitled to attend and vote at a general meeting shall be entitled to appoint another person (whether a shareholder or not) as his proxy to attend and vote instead of himself but a proxy so appointed shall not have any right to speak at the meeting. Section 3(2BBA)(a) A corresponding new bank may from time to time and after any paid-up capital him been raised by public issue under clause (c) of sub-Section (2B), by resolution passed at an annual general meeting of the shareholders entitled to vote, voting in person, or, where proxies are allowed, by proxy, and the votes cast in favour of the resolution are not less than three times the number of the votes, if any, cast against the resolution by the shareholders so entitled and voting, reduce its paid-up capital in any way. Section 3(2E) No shareholder of the corresponding new 153 bank, other than the Central Government, shall be entitled to exercise voting rights in respect of any shares held by him in excess of one per cent of the total voting rights of all the shareholders of the corresponding new bank. 91 To have calls on shares on a uniform Regulations 22, 24, 26 and 29 Regulation 22: Calls on shares basis on all the shares falling under the The Board may make such calls as it thinks same class. fit for all moneys remaining unpaid, which are by the conditions of allotment not made payable at fixed times, and each shareholder shall pay the amount of every call so made on him to the person and at the time and place appointed by the Board. 99 Right to determine that uncalled share No corresponding provision. capital shall be capable of being called only upon winding up. 106 To consent to the variation of rights No corresponding provision. attached to the shares. 107 To apply to the National Company Law No corresponding provision. Tribunal to have the variation of rights cancelled if no consent is given. 108 To transfer shares held in the company. Regulations 3, 17, 18 and 19 Regulation 3: Nature of Shares Section 3(2D) The shares of the bank shall be movable property, transferable in the manner provided in the bank Regulations which include a detailed procedure for such transfer Section 3(2D) The shares of every corresponding new bank not held by the central Government shall be freely transferable: Provided that no individual or company resident outside India or any company incorporated under any law not in force in India of any branch of such company, whether resident outside India or not, shall at any time hold or acquire by transfer or otherwise shares of the corresponding new bank so that such investment in aggregate exceed the percentage, not being more than twenty per cent of the paid-up capital as may be specified by the Central Government by notification in the Official Gazette. 154 110 To apply for the registration of transfer Regulations 18 and 19 Regulation 18: Power to suspend transfers of shares. The board or the committee designated by the board shall not register any transfer during any period in which the register is closed. Regulation 19: Board’s right to refuse registration of transfer of shares (i) The board or committee may refuse transfer of any shares in the name of the transferee on any one or more of the following grounds, and on no other grounds: a. the transfer of shares is in contravention of the provisions of the Bank Acquisition Act or regulations made thereunder or any other law or that any other requirement under the law relating to registration of such transfer has not been complied with; b. the transfer of shares, in the opinion of the board, is prejudicial to the interests of the bank or to public interest; c. the transfer of shares is prohibited by an order of court, tribunal or any other authority under any law for the time being in force; d. an individual or a company resident outside India or any company incorporated under any law not in force in India or any branch of such company, whether resident outside India or not, will on the transfer being allowed, hold or acquire as a result thereof, shares of the bank and such investment in the aggregate will exceed the percentage being more than 20% of the paid up capital or as may be prescribed by the central government by notification in the official gazette. (ii) The board or the committee shall, after the instrument of transfer of shares of the bank is lodged with it for the purpose of registration of such transfer, form its opinion as to whether such registration ought or ought not to be refused on any of the grounds referred to above: a. if it has formed the opinion that such registration ought not to be so refused, effect such registration; and 155 b. if it has formed the opinion that such registration ought to be refused on any of the grounds mentioned above intimate the same to the transferor and the transferee by notice in writing giving reasons for such refusal within 60 days from the receipt of the transfer form or within such period as may be laid down in the listing agreement with the concerned stock exchange. 111, To make a petition to the National No corresponding provision. 111A Company Law Tribunal against refusal by the company for the registration of transfer of shares or rectification of the register of members. 112 To have the share transfer instrument Regulation 16 Regulation 16: Consolidation and sub- certified by the company in the case of division of shares part transfer of the total holding. On a written application made by the shareholder(s), the board or the committee designated by it may consolidate or sub- divide the shares submitted to it for consolidation/ sub-division as the case may be and issue new certificate(s) in lieu thereof on payment to the bank of its costs, charges and expenses of and incidental to the matter. 113 To have the share certificate delivered Regulation 17(v) Regulation 17(v) within the time limit stipulated. Unless the transfer of shares is refused under regulation 19, the share certificate duly transferred shall be delivered to the transferee within sixty days of the date of lodging the instrument of transfer. 113 To serve on the company a notice, No corresponding provision. requiring it to make good any default in delivering share/ debenture/ stock certificates. 113 To apply to the Central Government in No corresponding provision. the event of the company’s failure to make good any default as above. 117A To inspect debenture trust deed and No corresponding provision. obtain a copy thereof. 156 118 To request the company to forward a No corresponding provision. copy of a debenture trust deed. 118 To apply to the Central Government to No corresponding provision. direct the company to forward a copy of the debenture trust deed forthwith. 134 To file with the Registrar of Companies No corresponding provision. the particulars of a charge created by the company, as a person interested therein. 141 To apply to the Central Government for No corresponding provision. the rectification of the register of charges as an interested party. 144 To inspect the copies of instruments No corresponding provision. creating charge. 144 To apply to the Central Government for No corresponding provision. compelling inspection of the copies and the register aforesaid, in case of refusal by the company. 163 To inspect, to obtain copies of the Regulations 11 and 12 Share register maintained under Regulation register of members or register of 5 and Section 3(2F) debenture holders and copies of annual Regulation 11: Inspection of Register returns together with the copies of certificates and documents required to The register shall, except when closed be annexed thereto. under Regulation 12, be open to inspection, free of charge, at the place where it is maintained during business hours, subject to such reasonable restrictions as the board may impose, but that not less than two hours in each working day be allowed for inspection Notwithstanding anything contained in sub- regulation (ii), any duly authorized officer of the Government shall have the right to make a copy of any entry in the register or be furnished a copy of the register or any part thereof. 163 To apply to the central Government for No corresponding provision. compelling inspection of the registers of shareholders and debenture-holders and annual returns. 165 To receive a copy of the statutory report No corresponding provision. and to attend the statutory meeting. 157 165 To discuss at the statutory meeting, any No corresponding provision. matter relating to the formation of the company or arising out of the statutory report, without giving a previous notice therefor. 165 To have accessible, a list of members No corresponding provision. with details, during the continuation of the statutory meeting. 169 To attend an extra-ordinary general Regulations 60 Regulation 60: Persons entitled to attend meeting. general meetings (i) All directors and all shareholders of the bank shall, subject to the provisions of sub- regulation (ii), be entitled to attend a general meeting. (ii) A shareholder (not being the central government) or a director, attending a general meeting shall for the purpose of identification and to determine his voting rights, be required to sign and deliver to the bank a form to be specified by the chairman containing the particulars relating to: (a) his full name and registered address; (b) the distinctive number of his shares; (c) whether he is entitled to vote and the number of votes to which he is entitled in person or by proxy or as a duly authorized representative. 169 To requisition an extra-ordinary general Regulation 57 Regulation 57: Extraordinary General meeting. Meeting (i) The chairman and managing director or in his absence the executive director of the bank or in his absence any one of the directors of the bank may convene an extra- ordinary general meeting or shareholders, if so directed by the board, or on a requisition for such a meeting having been received either from the central government or from other shareholders holding shares carrying, in the aggregate not less than ten percent of the total voting rights of all shareholders. 158 (ii) The requisition referred in sub-regulation (i) shall state the purpose for which the extra ordinary general meeting is required to be convened, but may consist of several documents in like form each signed by one or more of the requisitionists. (iii) Where two or more persons hold any shares jointly, the requisition or a notice calling a meeting signed by one or some of them shall, for the purpose of this regulation have the same force and affect as if it had been signed by all of them. (iv) The time, date and place of the extra ordinary general meeting shall be decided by the board: Provided that the extraordinary general meeting convened on the requisition by the central government or other shareholder shall be convened not later than 45 days of the receipt of the requisition. (v) If the chairman and managing director or in his absence the executive director, as the case may be, does not convene a meeting as required by sub-regulation (i), within the period stipulated in the proviso to sub- regulation (iv), the meeting may be called by the requisitionist themselves within three months from the date of the requisition: Provided that nothing in this sub-regulation shall be deemed to prevent a meeting duly convened before the expiry of the period of three months aforesaid, from being adjourned to some day after the expiry of that period. (vi) A meeting called under sub-regulation (v) by the requisitionist shall be called in the same manner, as nearly as possible as that in which the other general meetings are called by the board. 169 To hold an extra-ordinary general meeting Regulation 57 As above if the board of directors fails to convene a meeting on the requisition, within the time limit stipulated. 159 169 To have reimbursed, the expenses No corresponding provision. incurred for convening/ holding the extra-ordinary general meeting, on failure of the board as aforesaid. 171,172 To receive a notice of every general Regulation 56 Regulation 56: Notice convening an Annual meeting. General Meeting (i) A notice convening an annual general meeting of the shareholders shall be published at least twenty one clear days before the meeting in not less than two daily newspapers having wide circulation in India. (ii) The time and date of such meeting shall be as specified by the board. The meeting shall be held at the place of head office of the bank. 173 To have the notice of a general meeting No corresponding provision. annexed with an explanatory statement. 174 To be counted for the purpose of Regulation 58 Regulation 58: Quorum of general meeting constituting a quorum at a general No business shall be transacted at any meeting. meeting of the shareholders unless a quorum of at least five shareholders entitled to vote at such meeting in person are present at the commencement of such business 176 To appoint a proxy to attend and vote at Regulations 68 and 70 Shareholders can attend and vote a general meeting of a company. personally and through proxy 176 To inspect the proxies lodged with the No corresponding provision. company in the manner specified. 179 To demand a poll and to withdraw the Regulation 61 Regulation 61: Voting at general meetings demand in the manner laid down in this At any general meeting, a resolution put to section at a general meeting of the the vote of the meeting shall, unless a poll company. is demanded be decided on a show of hands. 182 Not to be prohibited from exercising the No corresponding provision. voting right on the ground that the shares or other interest in the company has not been held for any specified period or on any other ground than the one specified in s 181. 160 183 To use votes on a poll differently, for or No corresponding provision. against the resolution. 184 To be appointed as a scrutineer at a poll. Regulation 61A Regulation 61A: Scrutineers at a Poll Of the two scrutineers appointed under this regulation one shall always be a shareholder (not being an officer or employee of the bank) present at the meeting; provided that such a shareholder is available and willing to be appointed. 186 To apply to the National Company Law No corresponding provision. Tribunal to order a general meeting other than annual general meeting, to be called. 187 To be represented at a general meeting Regulation 69(i) Regulation 69(i): Voting by duly authorised of a company (if the member to be representative - represented, is a company). A shareholder, being the Central Government or a company, may authorise any of its officials or any other person to act as its representative at any general meeting. The authorisation so given may be in favour of two persons in the alternative and in such a case any one of such persons may act as the duly authorised representative of the Central Government / company. 187A To be represented at a general meeting Regulation 69(i) Same as of a company (if the member to be above represented, is the President or Governor of a State). 188 To have a resolution to be proposed at a No corresponding provision. general meeting to be circulated amongst members of the company. 190 To give a special notice to the company No corresponding provision. in respect of the resolutions requiring special notice. 190 To be given a notice of a resolution No corresponding provision. requiring special notice. 196 To inspect and be furnished with a copy Regulation 62 Regulation 62: Minutes of general of the minutes of any general meeting. meetings - (i) The bank shall cause the minutes of all 161 proceedings to be maintained in the books kept for the purpose. (ii) On written request made by a shareholder for inspection of the minute book or for a copy of the minute of a specified meeting, the bank shall allow the inspection or furnish the copy of the minute, as the case may be. 196 To apply to the central Government for No corresponding provision. inspection of the minutes books or to be furnished with a copy of minutes of the general meeting. 203 To apply to the court/ National Company No corresponding provision. Law Tribunal to restrain fraudulent persons from managing companies. 205A, To claim any unclaimed dividend. No corresponding provision. 205B 206 To receive dividend declared. No corresponding provision. 206A To have the dividend transferred to the No corresponding provision. special account where the transfer of shares has not been registered by the company. 206A To have any rights shares or bonus No corresponding provision. shares kept in abeyance in relation to the shares, the registration of transfer of which has not been registered by the company. 219 To receive 21 days before the date of No corresponding provision. the annual general meeting, copies of the balance sheet, profit and loss account, directors’ report, auditor’s report and other documents. 219 To require a company to furnish the full No corresponding provision. accounts where the company has sent abridged accounts. 219 To inspect the full accounts at the annual Section 10A Section 10A: Annual general meeting general meeting if the company has sent The shareholders present at an annual the abridged accounts. general meeting shall be entitled to discuss the balance sheet and the profit and loss 162 account of the corresponding new bank made up to the previous 31st day of March, the report of the board of directors on the working and activities of the corresponding new bank for the period covered by the accounts and the auditor’s report on the balance sheet and accounts. 224(5) To nominate a person for the No corresponding provision. appointment as auditor of the company if the first auditors are removed at a general meeting. 225 To give a special notice for a resolution No corresponding provision. appointing as auditor, person other that the retiring auditor or providing expressly that a retiring auditor shall not be reappointed. 227 To be reported upon the accounts, Section 30(2), Banking The auditor shall discharge the duties and balance sheet, profit and loss account, Regulation Act, 1949. be subject to the liabilities imposed on etc by the auditors of the company. auditors of companies by Section 227 of the Companies Act, 1956. This provision is applicable to corresponding new banks by virtue of the provisions of Section 51 of the Banking Regulation Act. 230 To inspect the auditors’ report at the Section 10 A general meeting where it is laid. 250 To apply to the National Company Law No corresponding provision. Tribunal to impose restrictions on shares, debentures or to prohibit transfer thereof in certain cases. 257 To stand for election for directorship at Regulation 63 Regulation 63: Directors to be elected at a general meeting. Section 9(3) general meeting - (i) A director under clause (i) of sub-Section (3) of Section 9 of the Act shall be elected by the shareholders on the register, other than the central Government, from amongst themselves in the general meeting of the bank. 257 To give notice to the company for Regulation 65 Regulation 65: Nomination of candidates for proposing a resolution at a general election meeting to have himself or any other (i) No nomination of a candidate for election person elected as a director. as a director shall be valid unless, 163 a. he is a shareholder holding not less than 100 (one hundred) shares in the bank; b. he is on the last date for receipt of nomination, not disqualified to be a director under the Act or under the Scheme; c. he has paid all calls in respect of the shares of the bank held by him, whether alone or jointly with others, on or before the last date fixed for the payment of the call; d. nomination is in writing signed by at least one hundred shareholders entitled to elect directors under the Act. (ii) No nomination shall be valid unless it is received with all the connected documents complete in all respects and received, at the head office of the bank on a working day not less than fourteen days before the date fixed for the meeting. 257 To be informed of the candidature for No corresponding provision. directorship or the intention of a member to propose such person for directorship. 265 To vote for appointing a director by the No corresponding provision. proportional representation system. 284 To give a notice to the company Clause 11A and Section 9(3B) Clause 11-A: Removal from office of an proposing a resolution at a general elected director meeting for removal of a director and The shareholders other than the Central appointment of a person as director in Government, may, by a resolution passed place of the removed director. by majority of the votes of such shareholders holding in the aggregate not less than one half of the share capital held by all such shareholders, remove any director elected by the shareholders and elect in his stead another person to fill the vacancy. 301 To inspect, to take copies of the register No corresponding provision. of contracts, etc kept under this Section. 302 To receive an abstract of the terms of No corresponding provision. the contract or variation thereof, in respect of the appointment of manager or managing director, in which any director is interested. 164 304 To inspect the register of directors. No corresponding provision. 304 To apply to the Central Government/ No corresponding provision. National Company Law Tribunal for compelling inspection of the aforesaid register, if it is refused by the company. To inspect the register kept by the registrar under this Section. 306 To inspect at the annual general meeting, No corresponding provision. the register of directors’ shareholdings. 307 To inspect at the registered office, the No corresponding provision. register of directors’ shareholdings. 307 To apply to the Central Government/ No corresponding provision. National Company Law Tribunal for compelling inspection of the aforesaid register, if refused by the company. 391 To apply to the National Company Law No corresponding provision. Tribunal to call a meeting of the creditors or of the members in case compromise or arrangement is proposed with its creditors or members. 391 To agree to any compromise or No corresponding provision. arrangement at a special meeting called for that purpose. 391 To have disclosed in the notice of the No corresponding provision. special meeting referred to above, the particulars notified in sub-s (1) of this Section. 393 To be furnished by the company, with a No corresponding provision. copy of the statement setting forth the terms of the compromise or arrangement proposed and explaining its effect where the notice of the meeting is given by an advertisement. 393 To be offered the same terms as offered No corresponding provision. to all holders of shares of that class, whose transfer is involved if the member is a dissenting member. 165 395 To receive notice within one month from No corresponding provision. the date of the transfer, of that fact in the prescribed manner, to the holders of the remaining shares or of the remaining shares of that class, as the case may be, who have not assented to the scheme or contract. 395 To require the transferee company to No corresponding provision. acquire the shares in question within three months of giving the notice. 395 To have a copy of the notice transmitted No corresponding provision. to the transferor company together with an instrument of transfer and within one month of the date of registration be informed of the fact of registration, and of the receipt of amount or other consideration. 395 To have a copy of the notice transmitted No corresponding provision. to the transferor company together with an instrument of transfer and within one month of the date of registration, be informed of the fact of registration and of the receipt of amount or other consideration. 396 To have as nearly as may be, the same No corresponding provision. interests in or rights against the company resulting from the amalgamation, as he had in the company in which he was originally a member, and to receive compensation in case such interests or rights are reduced. 396 If aggrieved by any assessment of No corresponding provision. compensation, to prefer an appeal to the National Company Law Tribunal. 397, 398 To apply to the National Company Law No corresponding provision. Tribunal if he is of the opinion that the affairs of the company are being conducted in a manner prejudicial to the public interest or in a manner oppressive to any member. 166 408 To apply to the National Company Law No corresponding provision. Tribunal for appointment of nominee director, to safeguard the interests of the company or its shareholders or the public interests. 433 To resolve along with other members, No corresponding provision. at a general meeting, that the company be wound-up by the National Company Law Tribunal. 439 To petition the National Company Law No corresponding provision. Tribunal for winding-up the company. 440 To present winding-up petition where No corresponding provision. company is being wound-up voluntarily or subject to court’s supervision. 490 To appoint one or more liquidators in the No corresponding provision. case of voluntary liquidation, and to fix the remuneration if any, to be paid to the liquidator. 492 To fill the vacancy occurred by the death, No corresponding provision. resignation or otherwise in the office of the liquidator appointed by the company, in a general meeting and convene a general meeting for this purpose. 511 To have distributed to himself on the No corresponding provision. winding-up, the assets of the company according to his rights and interests in the company. 545 To have the opportunity of making a No corresponding provision. statement in writing to the registrar and being heard thereon, in case he is being prosecuted as a delinquent member. 621 To make a complaint in a court regarding No corresponding provision. offence under the Companies Act. Please note the following for the above table; 1. All references to Sections are references to Sections of the Banking Companies (Acquisition & Transfer of Undertakings) Act, 1970, except where otherwise specified. 2. All references to Regulations are references to provisions of the Punjab National Bank (Shares and Meetings) Regulations, 2000 3. All references to Clauses are references to provisions of the Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970 167 Licensing of Corresponding New Banks Section 22 of the Banking Regulation Act, which requires a licence to be obtained from the RBI in order to carry out banking business in India, applies only to banking companies, and not corresponding new banks. Accordingly, the Punjab National Bank does not require a licence in order to carry out banking activities. Regulations relating to the Opening of Branches Banks are required to obtain licences from the RBI to open new branches. Permission is granted based on factors such as overall financial position of the bank, quality of its management, efficacy of the internal control system, profitability and other relevant factors. The RBI may cancel the licence for violations of the conditions under which it was granted. It is left to the judgment of the individual banks to assess the needs for opening additional branches. Capital Adequacy Requirements We are subject to the capital adequacy requirements of the RBI, which is based on the guidelines of the Basel Committee on Banking Regulations and Supervisory Practices, 1998. With a view to adopting the Basel Committee framework on capital adequacy norms which takes into account the elements of risk in various types of assets in the balance sheet as well as off- balance sheet business and also to strengthen the capital base of banks, RBI decided in April 1992 to introduce a risk asset ratio system for banks (including foreign banks) in India as a capital adequacy measure. This requires us to maintain a minimum ratio of capital to risk adjusted assets and off-balance sheet items of 9%, at least half of which must be Tier 1 capital. The total capital of a banking company and a corresponding new bank is classified into Tier 1 and Tier 2 capital. Tier 1 capital i.e. the core capital, provides the most permanent and readily available support against unexpected losses. It comprises paid-up capital and reserves consisting of any statutory reserves, free reserves and capital reserve as reduced by equity investments in subsidiaries, intangible assets, and losses in the current period and those brought forward from the previous period. A bank’s deferred tax asset is to be treated as an intangible asset and deducted from its Tier 1 capital. Tier 2 capital i.e. the undisclosed reserves and cumulative perpetual preference shares, revaluation reserves (at a discount of 55%), general provisions and loss reserves (allowed up to a maximum of 1.25% of risk weighted assets), hybrid debt capital instruments (which combine certain features of both equity and debt securities) and subordinated debt. Any subordinated debt is subject to progressive discounts each year for inclusion in Tier 2 capital and total subordinated debt considered as Tier 2 capital cannot exceed 50% of Tier 1 capital. With a view to the building up of adequate reserves to guard against any possible reversal of the interest rate environment in the future due to unexpected developments, the RBI has advised banks to build up an investment fluctuation reserve of a minimum of 5% of the bank’s investment portfolio within a period of five years from fiscal 2001. This reserve has to be computed with respect to investments in held for trading and available for sale categories. Investment fluctuation reserve is included in Tier 2 capital. Though investment fluctuation reserve is also considered general provision for Tier 2 but the same is not be subjected to the ceiling of 1.25% of risk weighted assets. Risk adjusted assets and off-balance sheet items considered for determining the capital adequacy ratio are the weighted aggregate of specified funded and non-funded exposures. Degrees of credit risk expressed as percentage weighting are assigned to various balance sheet asset items and conversion factors to off-balance sheet items. The value of each item is multiplied by the relevant weight or conversion factor to produce risk-adjusted values of assets and off-balance-sheet items. Guarantees and letters of credit are treated as similar to funded exposure and are subject to similar risk weight. All foreign exchange and gold open position limits carry a 100% risk weight. A risk weight of 2.50% to cover market risk has to be assigned in respect of the entire investments portfolio over and above the risk weight for credit risk. Banks are required to assign a 100% risk weight for all state government guaranteed securities issued by defaulting entities. The aggregate risk weighted assets are taken into account for determining the capital adequacy ratio. As per regulatory requirements, banks have to maintain a capital to risk asset ratio of 9%. However, as per RBI guidelines issued in September 2002, in addition to other conditions to be complied with for declaration of dividend without approval of RBI, capital to risk asset ratio must also be maintained at 11%. 168 Asset Classification and Provisioning In April 1992, the RBI issued formal guidelines on income recognition, asset classification, provisioning standards and valuation of investments applicable to banks, applicable from the financial year 1992-93, which are revised from time to time. As per these guidelines, the basis of treating various credit facilities as non-performing are set forth below. Non-Performing Assets An advance is a non-performing asset where: ● interest and/or installment of principal remained overdue for a period of more than 90 days in respect of a term loan; ● the account remained “out-of-order” for a period of more than 90 days in respect of an overdraft or cash credit (if the outstanding balance remains continuously in excess of the sanctioned limit/ drawing power or there are no credits continuously for 90 days as on the date of balance sheet or credits are not enough to cover the interest debited during the same period, then such accounts are treated as “out of order”.); ● the bill remained overdue for a period of more than 90 days in case of bills purchased and discounted; ● if the interest and/or principal remained overdue for two harvest seasons but for a period not exceeding two half-years in the case of an advance granted for agricultural purposes. With effect from September 30, 2004, a loan granted for short duration crops will be treated as a non performing asset, if the installment of principal or interest thereon remains overdue for two crop seasons. With effect from September 30, 2004, a loan granted for long duration crops will be treated as a non performing asset, if the installment of principal or interest thereon remains overdue for one crop season. (Crops with crop season longer than one year are long duration crops, and crops which are not long duration crops are treated as short duration crops.) ● any amount to be received remains overdue for a period of more than 90 days in respect of other accounts. Once the account has been classified as a non-performing asset, the unrealized interest and other income already debited to the account is derecognised and further interest is not recognised or credited to the income account unless collected. Asset Classification Non-performing assets are classified as described below: ● Sub-Standard Assets. Assets that are non-performing assets for a period not exceeding 18 months. With effect from 31 March 2005, a sub-standard asset would be one, which has remained NPA for a period less than or equal to 12 months. ● Doubtful Assets. Assets that are non-performing assets for more than 18 months. With effect from March 31, 2005, an asset will be classified as doubtful if it remains in the sub-standard category for 12 months. ● Loss Assets. Assets on which losses have been identified by the bank or internal or external auditors or the RBI inspection but the amount has not been written off fully. There are separate guidelines for projects under implementation which are based on the achievement of financial closure and the date of approval of the project financing. The RBI has separate guidelines for restructured assets under the corporate debt restructuring mechanism and under other mechanisms. A fully secured standard asset can be restructured by reschedulement of principal repayments and/ or the interest element, but must be separately disclosed as a restructured asset. The amount of sacrifice, if any, in the element of interest, measured in present value terms, is either written off or provision is made to the extent of the sacrifice involved. Similar guidelines apply to sub-standard assets, and to doubtful assets, in the case of restructuring of assets under the corporate debt restructuring mechanism. The sub-standard accounts which have been subjected to restructuring, whether in respect of principal instalment or interest amount, are eligible to be upgraded to the standard category only after the specified period, i.e., a period of one year after the date when first payment of interest or of principal, whichever is earlier, falls due, subject to satisfactory performance during the period. Provisioning and Write-Offs Provisions are based on guidelines specific to the classification of the assets. The following guidelines apply to the various 169 asset classifications: ● Standard Assets. A general provision of 0.25% is required. ● Sub-Standard Assets. A general provision of 10% on total outstanding should be made. The unsecured exposures which are identified as sub-standard would attract additional provision of 10%, i.e., a total of 20% on the outstanding balance. ● Doubtful Assets. A 100% provision/ write-off of the unsecured portion of advances which are not covered by realizable value of the security. In cases where there is a secured portion of the asset, depending upon the period for which the asset remains doubtful, a 20% to 100% provision is required to be made against the secured asset as follows: ● Up to one year: 20% provision ● One to three years: 30% provision ● More than three years: 1. In respect of outstanding stock of non performing assets as on March 31, 2004: 50% provision, which will become 60% with effect from March 31, 2005, 75% with effect from March 31, 2006 and 100% with effect from March 31, 2007. 2. In respect of assets, which have been doubtful for over three years on or after April 1, 2004, provision is to be raised to 100% with effect from March 31, 2005. ● Loss Assets. The entire asset is required to be written off or provided for. While the provisions indicated above are mandatory, banks are encouraged to make higher provisions over and above the mandatory level. Regulations relating to making loans The provisions of the Banking Regulation Act govern the making of loans by banks in India. The RBI issues directions covering the loan activities of banks. Some of the major guidelines of RBI, which are now in effect, are as follows: ● The RBI has prescribed norms for bank lending to non-bank financial companies and financing of public sector disinvestment. ● The banks should charge interest on loans/advances/cash credits/overdrafts or any other financial asisstance granted/ provided/renewed by them or discount usance bills in accordance with the directives on interest rates on advances issued by RBI from time to time. Banks are free to determine their own lending rates but each bank must declare its benchmark prime lending rate as approved by its board of directors. Benchmark prime lending rate is determined on the basis of various parameters, which inter alia, include cost of funds, operating expenses, capital charge and profit margin. Each bank should also indicate the maximum spread over the benchmark prime lending rate for all credit exposures other than retail loans over Rs. 200,000. The interest charged by banks on advances up to Rs. 200,000 to any one entity (other than most retail loans) must not exceed the benchmark prime lending rate. Banks are also given freedom to lend at a rate below the prime lending rate in respect of creditworthy borrowers and exporters on the basis of a transparent and objective policy approved by their boards. Interest rates for certain categories of advances are regulated by the RBI. Banks are also free to stipulate lending rates without reference to their own benchmark prime lending rates in respect of certain specified categories of loans. ● In terms of Section 20(1) of the Banking Regulation Act, a bank cannot grant any loans and advances on the security of its own shares. A bank is also prohibited from entering into any commitment for granting any loans or advances to or on behalf of any of its directors, or any firm in which any of its directors is interested as partner, manager, employee or guarantor, or any company (not being a subsidiary of the banking company or a company registered under Section 25 of the Companies Act, or a Government company) of which, or the subsidiary or the holding company of which any of the directors of the bank is a director, managing agent, manager, employee or guarantor or in which he holds substantial interest, or any individual in respect of whom any of its directors is a partner or guarantor. There are certain exemptions in this regard as the explanation to the Section provides that ‘loans or advances’ shall not include any transaction which the RBI may specify by general or special order as not being a loan or advance for the purpose of such Section. We are in compliance with these requirements. Legislation introduced in the Indian Parliament to amend the Banking Regulation Act has proposed to prohibit lending to 170 relatives of directors and to non-subsidiary companies that are under the same management as the banking company, joint ventures, associates or the holding company of the banking company. There are guidelines on loans secured by shares, debentures and bonds, money market mutual funds, gold/silver jewellery, etc. in respect of amount, margin requirement and purpose. Regulations relating to sale of assets to Asset Reconstruction Companies The Securitisation Act provides for sale of financial assets by banks and financial institutions to asset reconstruction companies. The RBI has issued guidelines to banks on the process to be followed for sales of financial assets to asset reconstruction companies. These guidelines provide that a bank may sell financial assets to an asset reconstruction company provided the asset is a non-performing asset. A bank may sell a standard asset only if the borrower has a consortium or multiple banking arrangement, at least 75% by value of the total loans to the borrower are classified as non-performing and at least 75% by value of the banks and financial institutions in the consortium or multiple banking arrangement agree to the sale. The banks selling financial assets should ensure that there is no known liability devolving on them and that they do not assume any operational, legal or any other type of risks relating to the financial assets sold. Further, banks may not sell financial assets at a contingent price with an agreement to bear a part of the shortfall on ultimate realisation. However, banks may sell specific financial assets with an agreement to share in any surplus realised by the asset reconstruction company in the future. While each bank is required to make its own assessment of the value offered in the sale before accepting or rejecting an offer for purchase of financial assets by an asset reconstruction company, in consortium or multiple banking arrangements where more than 75% by value of the banks or financial institutions accept the offer, the remaining banks or financial institutions are obliged to accept the offer. Consideration for the sale may be in the form of cash, bonds or debentures or security receipts or pass through certificates issued by the asset reconstruction company or trusts set up by it to acquire the financial assets. Any loss on sale must be charged to the profit and loss account, but any gains must be used for meeting losses on sale of other financial assets. For computing capital adequacy, a risk weight of 102.50% is applied to instruments received by banks as consideration for sale of financial assets to asset reconstruction companies. Directed Lending Priority Sector Lending The RBI requires commercial banks to lend a certain percentage of their net bank credit to specific sectors (known as priority sectors), such as agriculture, small-scale industry, small businesses and housing finance. Total priority sector advances should be 40% of net bank credit with agricultural advances required to be 18% of net bank credit and advances to weaker sections required to be 10% of net bank credit, and 1% of the previous year’s net bank credit required to be lent under the Differential Rate of Interest scheme. Domestic scheduled commercial banks having shortfall in lending to priority sector are allocated amounts for contribution to the Rural Infrastructure Development Fund established in National Board for Agricultural and Rural Development. Any shortfall by foreign banks in the amount required to be lent to the priority sectors may be required to be deposited with the Small Industries Development Bank of India. The RBI requires banks to make advances towards housing finance. This can be in the form of home loans to individuals or subscription to the debentures and bonds of the National Housing Bank and housing development institutions recognised by the Government of India. The RBI also periodically issues instructions/directives to banks with regard to providing credit facilities to minority communities. Export Credit The RBI also requires commercial banks to make loans to exporters at concessional rates of interest. This enables exporters to have access to an internationally competitive financing option. Pursuant to existing guidelines, 12% of a bank’s net bank credit is required to be in the form of export credit. We provide export credit for pre-shipment and post-shipment requirements of exporter borrowers in rupees and foreign currencies. Credit Exposure Limits As a prudent measure aimed at better risk management and avoidance of concentration of credit risk, the RBI has prescribed credit exposure limits for banks and long-term lending institutions in respect of their lending to individual borrowers and to all 171 companies in a single group (or sponsor group). The limits set by the RBI are as follows: ● Credit exposure ceiling for a single borrower shall not exceed 15% of capital funds. Group exposure limit is 40% of capital funds. In case of financing for infrastructure projects, the single borrower exposure limit is extendable by another 5%, i.e., up to 20% of capital funds and the group exposure limit is extendable by another 10%, i.e. up to 50% of capital funds. Capital funds is the total capital as defined under capital adequacy standards (Tier 1 and Tier 2 capital). ● A bank may, in exceptional circumstances, with the approval of its board of directors, consider enhancement of the exposure over the above specified limits, up to a further 5% of capital funds. Punjab National Bank has decided that such enhancement may be considered for public sector undertaking borrowers based on their cash flows, and for non-public sector undertaking borrowers if they have a risk rating of ‘AA’ and above. ● Exposure shall include credit exposure (funded and non-funded credit limits) and investment exposure (including underwriting and other similar commitments) as well as certain types of investments in companies. The sanctioned limits or outstandings, whichever are higher, are considered for arriving at the exposure limit. Non-fund exposures are to be reckoned at 100% of the limit or outstandings, whichever is higher. Credit exposure is the aggregate of: ● all types of funded and non-funded credit limits; ● facilities extended by way of equipment leasing, hire purchase finance and factoring services; ● advances against shares, debentures, bonds and units of mutual funds to stock brokers and market makers; ● bank loan for financing promoters’ contributions; ● bridge loans against equity flows/issues; and ● financing of initial public offerings. Investment exposure comprises of the following elements: ● investments in shares and debentures of companies acquired through direct subscription, devolvement arising out of underwriting obligations or purchase from secondary markets or on conversion of debt into equity; ● investment in public sector undertaking bonds through direct subscription, devolvement arising out of underwriting obligations or purchase made in the secondary market; ● investments in commercial papers issued by corporate bodies or public sector undertakings; and ● investments in debentures, bonds, security receipts, pass through certificates issued by a securitisation or reconstruction company. However, initially, since only a few securitisation and reconstruction companies are being set up, banks will be allowed to exceed prudential exposure on account of such investments on a case-to-case basis. To ensure that exposures are evenly distributed, the RBI requires banks to fix internal limits of exposure to specific sectors. These limits are subject to periodic review by the banks. Regulations relating to Investments and Capital Market Exposure Limits There are no limits on the amount of investments by banks in non-convertible debt instruments. However, credit exposure limits specified by the RBI in respect of lending to individual borrowers and borrower groups also apply in respect of these investments. Pursuant to the RBI guidelines, the exposure of banks to capital markets by way of investments in shares, convertible debentures, units of equity-oriented mutual funds and loans to brokers, should not exceed 5% of outstanding domestic advances (excluding inter-bank lending and advances outside India and including commercial paper) at March 31 of the previous fiscal year and investments in shares, convertible debentures and units of equity-oriented mutual funds should not exceed 20% of the bank’s net worth. In April 1999, the RBI, in its monetary and credit policy, stated that the investment by a bank in subordinated debt instruments, representing Tier 2 capital, issued by other banks and financial institutions should not exceed 10% of the investing bank’s capital including Tier 2 capital and free reserves. 172 In December 2003, the RBI issued guidelines on investments by banks in non-Statutory Liquidity Ratio securities issued by companies, banks, financial institutions, central and state government sponsored institutions and special purpose vehicles. These guidelines apply to primary market subscriptions and secondary market purchases. Pursuant to these guidelines, banks are prohibited from investing in non-Statutory Liquidity Ratio securities with an original maturity of less than one year, other than commercial paper and certificates of deposits. Banks are also prohibited from investing in unrated securities. A bank’s investment in unlisted non-Statutory Liquidity Ratio securities may not exceed 10% of its total investment in non-Statutory Liquidity Ratio securities as at the end of the preceding fiscal year. These guidelines will not apply to investments in security receipts issued by securitisation or reconstruction companies registered with the RBI and asset backed securities and mortgage backed securities with a minimum investment grade credit rating. These guidelines have been effective from April 1, 2004, with provision for compliance in a phased manner by January 1, 2005. Consolidated Supervision Guidelines In fiscal 2003, the RBI issued guidelines for consolidated accounting and consolidated supervision for banks. These guidelines became effective April 1, 2003. Punjab National Bank adopted these guidelines on April 1, 2002. The principal features of these guidelines are: ● Banks are required to prepare consolidated financial statements intended for public disclosure. ● Banks are required to submit to the RBI, consolidated prudential returns reporting their compliance with various prudential norms on a consolidated basis, excluding insurance subsidiaries. Compliance on a consolidated basis is required in respect of the following main prudential norms: - Single borrower exposure limit of 15% of capital funds (20% of capital funds provided the additional exposure of up to 5% is for the purpose of financing infrastructure projects); - Borrower group exposure limit of 40% of capital funds (50% of capital funds provided the additional exposure of up to 10% is for the purpose of financing infrastructure projects); - Deduction from Tier 1 capital of the bank, of any shortfall in capital adequacy of a subsidiary for which capital adequacy norms are specified; and - Consolidated capital market exposure limit of 2% of total on-balance sheet assets (excluding intangible assets and accumulated assets). Within the total limit, investment in shares, convertible bonds and debentures and units of equity oriented mutual funds should not exceed 10% of the bank’s consolidated net worth. Banks’ Investment Classification and Valuation Norms The salient features of the RBI’s guidelines on investment classification and valuation are given below: ● The entire investment portfolio is required to be classified under three categories: (a) held to maturity, (b) held for trading and (c) available for sale. Banks should decide the category of investment at the time of acquisition. ● Held to maturity investments compulsorily include (a) recapitalisation bonds, (b) investments in subsidiaries and joint ventures and (c) investments in debentures deemed as advance. Held to maturity investments also include any other investment identified for inclusion in this category subject to the condition that such investments cannot exceed 25% of the total investment excluding recapitalisation bonds and debentures. ● Profit on sale of investments in this category should be first taken to the profit and loss account and thereafter be appropriated to the capital reserve account. Loss on sale will be recognised in the profit and loss account. ● Investments under the held for trading category should be sold within 90 days; in the event of inability to sell due to adverse factors including tight liquidity, extreme volatility or a unidirectional movement in the market, the unsold securities should be shifted to the available for sale category. ● Profit or loss on the sale of investments in both held for trading and available for sale categories is taken in the profit and loss account. ● Shifting of investments from or to held to maturity may be done with the approval of the board of directors once a year, normally at the beginning of the accounting year; shifting of investments from available for sale to held for trading may be done with the approval of the board of directors, the asset liability management committee or the investment committee; shifting from held for trading to available for sale is generally not permitted. 173 Held to maturity securities are not marked to market and are carried at acquisition cost or at an amortized cost if acquired at a premium over the face value. Securities classified as available for sale or held for trading are valued at market or fair value as at the balance sheet date. Depreciation or appreciation for each basket within the available for sale and held for trading categories is aggregated. Net appreciation in each basket, if any, that is not realised is ignored, while net depreciation is provided for. Investments in security receipts or pass through certificates issued by asset reconstruction companies or trusts set up by asset reconstruction companies should be valued at the lower of the redemption value of the security receipts / pass-through certificates, and the net book value of the financial asset. Restrictions on Investments in a Single Company No bank may hold shares in any company, whether as owner or as pledge or mortgagee, exceeding the lower of 30% of the paid up share capital of that company and 30% of its own paid up share capital and reserves, whichever is less, except as statutorily provided. Limit on Transactions through Individual Brokers Guidelines issued by the RBI require banks to empanel brokers for transactions in securities. These guidelines also require that a disproportionate part of the bank’s business should not be transacted only through one broker or a few brokers. The RBI specifies that not more than 5% of the total transactions in securities through empanelled brokers can be transacted through one broker. If for any reason this limit is breached, the RBI has stipulated that the board of directors of the bank concerned should be informed on a half-yearly basis of such occurrences. Prohibition on Short-Selling The RBI does not permit short selling of securities by banks. Regulations Relating to Deposits The RBI has permitted banks to independently determine rates of interest offered on term deposits. Primary (urban) co- operative banks are permitted to pay interest on current account deposits at rates not exceeding 0.50% per annum. Further, banks may only pay interest of 3.50% per annum on savings deposits. In respect of savings and time deposits accepted from employees, we are permitted by the RBI to pay an additional interest of 1% over the interest payable on deposits from the public. Domestic term deposits have a minimum maturity of 7 days and a maximum maturity of 10 years. Time deposits from NRIs denominated in foreign currency have a minimum maturity of one year and a maximum maturity of three years. The RBI has permitted banks the flexibility to offer varying rates of interests on domestic deposits of the same maturity subject to the following conditions: ● Time deposits are of Rs. 1.50 million and above; and ● Interest on deposits is paid in accordance with the schedule of interest rates disclosed in advance by the bank and not pursuant to negotiation between the depositor and the bank. The RBI has stipulated that the interest rate on NRE deposits accepted before July 17, 2003 should not exceed 250 basis points and interest rates on those NRE deposits accepted before September 15, 2003 should not exceed 100 basis points over the US dollar LIBOR/ swap rates for the corresponding maturity. Further, NRE deposits contracted effective close of business in India on October 18, 2003 should not exceed 25 basis points over the US dollar LIBOR/ swap rates for the corresponding maturity, and those contracted effective close of business in India on April 17, 2004 should not exceed the LIBOR/ swap rates for US dollar of corresponding maturity. 174 Deposit Insurance Demand and time deposits of up to Rs. 100,000 accepted by Indian banks have to be compulsorily insured with the Deposit Insurance and Credit Guarantee Corporation, a wholly-owned subsidiary of the RBI. Banks are required to pay the insurance premium for the eligible amount to the Deposit Insurance and Credit Guarantee Corporation on a semi-annual basis. The cost of the insurance premium cannot be passed on to the customer. Regulations relating to Knowing the Customer and Anti-Money Laundering The RBI has issued several guidelines relating to identification of depositors and has advised banks to put in place systems and procedures to control financial frauds, identify money laundering and suspicious activities, and monitor high value cash transactions. The RBI has also issued guidelines from time to time advising banks to be vigilant while opening accounts for new customers to prevent misuse of the banking system for perpetration of frauds. The RBI requires banks to open accounts only after verifying the identity of customers as to their name, residence and other details to ensure that the customer is opening the account in his own name. To open an account, a prospective customer is required to be introduced by an existing customer who has had his own account with the bank for at least six months and has satisfactorily conducted that account, or a well-known person in the local area where the prospective customer resides. If the prospective customer does not have an introducer, the prospective customer is required to submit documents such as his identity card, passport or details of bank accounts with other banks. It must be made incumbent upon him to provide sufficient proof of his antecedents before the account is allowed to be opened. The Prevention of Money Laundering Act, 2002 has been passed by Indian Parliament and has received the assent of the President of India on January 17, 2003. The Act seeks to prevent money laundering and to provide for confiscation of property derived from, or involved in, money laundering and for incidental and connected matters. Legal Reserve Requirements Cash Reserve Ratio A corresponding new bank such as us is required to maintain a specified percentage of its demand and time liabilities, excluding inter-bank deposits, by way of balance in current account with the RBI. The cash reserve ratio can be a minimum of 3% and a maximum of 20% pursuant to Section 42 of the Reserve Bank of India Act, 1934. In September 18, 2004, the cash reserve ratio was made 4.75%. From October 2, 2004, it has been increased to 5%. Paid up capital, reserves, credit balance in the profit and loss account of the bank, amount availed of as refinance from the RBI, and apex financial institutions, provision for income tax in excess of the actual estimated liabilities, specified inter bank term deposits/term borrowing liabilities are excluded from the calculation of the cash reserve ratio: The RBI pays no interest on the cash reserves up to 3% of the demand and time liabilities and pays interest on the eligible cash balances, currently at the rate of 3.50%. Earlier, interest was paid by the RBI at the bank rate. The cash reserve ratio has to be maintained on an average basis for a fortnightly period and should not be below 70% of the required cash reserve ratio on any day of the fortnight. Statutory Liquidity Ratio In addition to the cash reserve ratio, a corresponding new bank such as us is required to maintain a specified minimum percentage of its net demand and time liabilities by way of liquid assets like cash, gold or approved securities. The percentage of this liquidity ratio is fixed by the RBI from time to time, and it can be a minimum of 25% and a maximum of 40% pursuant to Section 24 of the Banking Regulation Act. At present, the RBI requires banking companies to maintain a liquidity ratio of 25%. The Banking Regulation (Amendment) and Miscellaneous Provisions Bill, 2003 introduced in the Indian Parliament proposed to amend Section 24 of the Banking Regulation Act to remove the minimum Statutory Liquidity Ratio stipulation, thereby giving the RBI the freedom to fix the Statutory Liquidity Ratio below this level. 175 Regulations on Asset Liability Management At present, the RBI’s regulations for asset liability management require banks to draw up asset-liability gap statements separately for rupee and for four major foreign currencies. These gap statements are prepared by scheduling all assets and liabilities according to the stated and anticipated re-pricing date, or maturity date and behaviour studies that may be conducted by banks. These statements have to be submitted to the RBI on a monthly basis. The RBI has advised banks to actively monitor the difference in the amount of assets and liabilities maturing or being re-priced in a particular period and place internal prudential limits on the gaps in each time period, as a risk control mechanism. Additionally, the RBI has asked banks to manage their asset- liability structure such that the negative liquidity gap in the 1-14 day and 15–28 day time periods does not exceed 20% of cash outflows in these time periods. This 20% limit on negative gaps was made mandatory with effect from April 1, 2000. The Board of Directors has approved of these limits. In respect of other time periods, our Bank, on the basis of the RBI’s direction has laid down internal norms in respect of cumulative negative liquidity gaps. In case of interest rate sensitivity, our Bank has set limits on the maximum permissible impact on the net interest income during the one year period, due to a general change in the interest rates. Foreign Currency Dealership The RBI has granted us a full-fledged authorised dealers’ licence to deal in foreign exchange through our designated branches. Under this licence, we have been granted permission to: ● engage in foreign exchange transactions in all currencies; ● open and maintain foreign currency accounts abroad; ● raise foreign currency and rupee denominated deposits from NRIs; ● grant foreign currency loans to on-shore and off-shore corporations; ● open documentary credits; ● grant import and export loans; ● handle collection of bills, funds transfer services; ● issue guarantees; and ● enter into derivative transactions and risk management activities that are incidental to our normal functions authorised under our organizational documents. Our foreign exchange operations are subject to the guidelines specified by the RBI under the Foreign Exchange (Management) Act, 1999. As an authorised dealer, we are required to enroll as a member of the Foreign Exchange Dealers Association of India, which prescribes the rules relating to foreign exchange business in India. Authorised dealers, like us, are required to determine their limits on open positions and maturity gaps in accordance with the RBI’s guidelines and these limits are approved by the RBI. Further, we are permitted to hedge foreign currency loan exposures of Indian corporations in the form of interest rate swaps, options, currency swaps and forward rate agreements, subject to certain conditions. Statutes Governing Foreign Exchange and Cross-Border Business Transactions The foreign exchange and cross border transactions undertaken by banks are subject to the provisions of the Foreign Exchange Management Act. All branches should monitor all non-resident accounts to prevent money laundering. Restriction on Transfer of Shares Foreign investment in our Bank, as a corresponding new bank, is regulated by the provisions of the Bank Acquisition Act. Under Section 3(2D), foreign investment in new corresponding banks is subject to an overall statutory limit of 20% of the paid up capital. For public sector banks the RBI monitors the ceilings on Non Resident investments on a daily basis. For effective monitoring the RBI has fixed cut off points lower than the actual ceilings, which is 18% for public sector banks. Once the aggregate net purchases of equity shares of the respective bank by FIIs/NRIs/PIOs reaches the cut off points, which is 2% below the overall limit of 20%, the RBI cautions all designated bank branches so as not to purchase any more equity shares in the respective bank on behalf of NRIs/PIOs without prior approval of the RBI. The link offices are then required to intimate the 176 RBI about the total number and value of equity shares/convertible debentures of the bank they propose to buy on behalf of FIIs/ NRIs/PIOs. On receipt of such proposals, the RBI gives clearances on a first-come first-serve basis till such investment in banks reach the applicable sectoral caps/statutory ceilings. On reaching the aggregate ceiling limit, the RBI advises all designated bank branches to stop purchases on behalf of their FIIs/NRIs/PIOs clients. In addition, the provisions of the SEBI Takeover Regulations apply and must be complied with. Prohibited Business The Banking Regulation Act specifies the business activities in which a bank may engage. Banks are prohibited from engaging in business activities other than the specified activities. Reserve Fund Any bank incorporated in India is required to create a reserve fund to which it must transfer not less than 25% of the profits of each year before dividends. If there is an appropriation from this account, the bank is required to report the same to the RBI within 21 days, explaining the circumstances leading to such appropriation. The Government of India may, on the recommendation of the RBI, exempt a bank from the requirements relating to reserve fund. Restrictions on Payment of Dividends Pursuant to the provisions of the Banking Regulation Act, a bank can pay dividends on its shares only after all its capitalised expenses (including preliminary expenses, organization expenses, share selling commission, brokerage, amounts of losses and any other item of expenditure not represented by tangible assets) have been completely written off. The GoI may exempt banks from this provision by issuing a notification on the recommendation of the RBI. Currently, we have an exemption from the applicability of these requirements granted to us by the Government vide its notification bearing number F.No. 11/29/2004- BOA dated January 10, 2005. We require to secure exemptions from the MoF, GoI from the provisions of the Banking Regulation Act for payment of dividend. Prior approval of the RBI is required for a dividend payment above 25% of face value of a company’s shares or for an interim dividend payment. Further, as per RBI guidelines on payment of dividend, only those banks which comply with the following minimum prudential requirements are eligible to declare dividend with the prior approval of the RBI: ● Capital to risk asset ratio of at least 11% for the preceding two accounting years and for the accounting year for which it proposes to declare dividend. ● Net non performing assets of less than 3%. ● The dividend pay out ratio does not exceed 33.33%. ● The proposed dividend is payable out of the current year’s ratio. ● The financial statements pertaining to the financial year for which the bank is declaring a dividend should be free of any qualifications by the statutory auditors, which have an adverse bearing on the profit during that year. In the event that we fulfill the conditions stated above we can declare dividends without the consent of the RBI, but if we do not comply with the conditions stated above but wish to declare dividend or a higher rate of dividend we would require prior permission from the RBI. RBI has also notified that banks may also declare and pay interim dividends out of the relevant account period’s profit without the prior approval of the RBI if they satisfy the minimum criteria above, and the cumulative interim dividend is within the prudential cap on dividend payout ratio (33.33%) computed for the relevant accounting period. Declaration and payment of interim dividend beyond this limit would require the prior approval of the RBI. Restriction on Share Capital and Voting Rights Public sector banks can issue equity shares as per the SEBI Guidelines. The paid up capital of corresponding new banks may be increased by such amounts as the board of directors of the corresponding new bank may, after consultation with the RBI and with the previous sanction of the Central Government, raise by public issue of shares in such manner as may be prescribed, so however that the Central Government’s shareholding does not fall below 177 51% of the paid up capital of the bank. It may however be noted that the Banking Companies (Acquisition And Transfer Of Undertakings) And Financial Institutions Laws (Amendment) Bill, 2000 contemplates that the shareholding of the Central Government in corresponding new banks may not be less than 33% of the paid up capital. However, the bill has not yet been promulgated as an Act of the legislature and is not in force as on the date hereof. No shareholder of the corresponding new bank, other than the Central Government, shall be entitled to exercise voting rights in respect of any shares held by them in excess of one percent of the total voting rights of all the shareholders of the corresponding new bank. Regulatory Reporting and Examination Procedures The RBI is empowered under Section 27(2) of the Banking Regulation Act to inspect a bank. In 1995, RBI introduced a system of off-site monitoring and surveillance, with the primary objective of monitoring the financial condition of banks in between on- site examinations. The RBI monitors prudential parameters at quarterly intervals. To this end and to enable off-site monitoring and surveillance by the RBI, banks are required to report to the RBI on aspects such as: ● assets, liabilities and off-balance sheet exposures; ● risk weighting of these exposures, the capital base and the capital adequacy ratio; ● unaudited operating results for each quarter; ● asset quality; ● concentration of exposures; ● connected and related lending and the profile of ownership, control and management; ● ownership, control and management; ● structural liquidity and interest rate sensitivity; ● subsidiaries, associates and joint ventures; ● consolidated accounts and related financial information; ● information on risk based supervision; ● analysis of balance sheet; and ● other prudential parameters. The RBI also conducts periodic on-site inspections on matters relating to the bank’s portfolio, risk management systems, internal controls, credit allocation and regulatory compliance, at intervals ranging from one to three years. We are subject to the on-site inspection by the RBI at yearly intervals. The inspection report, along with the report on actions taken by us, has to be placed before our Board of Directors. On approval by our Board of Directors, we are required to submit the report on actions taken by us to the RBI. The RBI also discusses the report with the management team including the chief managing director and the chief executive director. The RBI also conducts on-site supervision of zonal branches, regional offices and other selected branches with respect to their general operations and foreign exchange related transactions. Appointment and Remuneration of the Chairman and Managing Director and Other Directors Directors on the Board of Directors of our Bank are appointed by the Central Government in terms of Section 9 of the Bank Acquisition Act. Further, a specified number of directors are appointed by the shareholders. The chairman and managing director are appointed by the Central Government in consultation with the RBI. The other directors appointed by the Central Government include an officer of the Reserve Bank appointed in consultation with the RBI and one employee and one workman director. The whole time directors to be appointed by the Central Government and the official of the Central Government who serves as the nominee director of the Central Government cannot be a director of any other corresponding new bank. For the text of Section 9 of the Bank Acquisition Act see the section titled “Main Provisions of Constitutional Documents” on page 248. The remuneration paid to directors is determined by the Central Government in consultation with the RBI. 178 Penalties The RBI may impose penalties on banks and their employees in case of infringement of regulations under the Banking Regulation Act. The penalty may be a fixed amount or may be related to the amount involved in any contravention of the regulations. The penalty may also include imprisonment. Assets to be Maintained in India Every bank is required to ensure that its assets in India (including import-export bills drawn in India and RBI approved securities, even if the bills and the securities are held outside India) are not less than 75% of its demand and time liabilities in India. Subsidiaries and other investments A bank requires the prior permission of RBI to incorporate a subsidiary. A bank is required to maintain an “arms’ length” relationship in respect of its subsidiaries and in respect of mutual funds sponsored by it in regard to business parameters such as taking undue advantage in borrowing/lending funds, transferring/selling/buying of securities at rates other than market rates, giving special consideration for securities transactions, in supporting/financing the subsidiary and financing our clients thro ugh them when we ourselves are not able or are not permitted to do so. Restriction on Creation of Floating Charge Prior approval of the RBI is required for creating floating charge on our undertaking or our property. Currently, all our borrowings including bonds are unsecured. Maintenance of Records We are required to maintain books, records and registers. The Banking Companies (Period of Preservation of Records) Rules, 1985 require a bank to retain records of books, accounts and other documents relating to stock and share register for a period of three years. Secrecy Obligations Under Section 13 of the Bank Acquisition Act, our Bank is statutorily bound to maintain secrecy about the affairs of its constituents, except in circumstances in which it is, in accordance with law or practices and usages customary among bankers, necessary or appropriate for the bank to divulge such information. Our obligations relating to maintaining secrecy arise out of common law principles governing our relationship with our customers. We cannot disclose any information to third parties except under clearly defined circumstances. The following are the exceptions to this general rule: ● where disclosure is required to be made under any law; ● where there is an obligation to disclose to the public; ● where we need to disclose information in our interest; and ● where disclosure is made with the express or implied consent of the customer. We are required to comply with the above in furnishing any information to any parties. We are also required to disclose information if ordered to do so by a court. The RBI may, in the public interest, publish the information obtained from the bank. Under the provisions of the Banker’s Books Evidence Act, 1891 a copy of any entry in a bankers’ book, such as ledgers, day books, cash books and account books certified by an officer of the bank may be treated as prima facie evidence of the transaction in any legal proceedings. The RBI has directed banks to incorporate consent clauses in loan agreements to enable disclosure of borrower information to credit bureaus. Regulations governing Offshore Banking Units The Government and the RBI have permitted banks to set up offshore banking units in special economic zones, which are specially delineated duty free enclaves deemed to be foreign territory for the purpose of trade operations, duties and tariffs. We have an offshore banking unit located in the Santacruz Electronic Exports Promotion Zone, Mumbai. The key regulations 179 applicable to offshore bank units include, but are not limited to, the following: ● No separate assigned capital is required. However, the parent bank is required to provide a minimum of US$ 10 million to its offshore banking unit. ● Offshore banking units are exempt from cash reserve ratio requirements. ● The RBI may exempt a bank’s offshore banking unit from statutory liquidity ratio requirements on specific application by the bank. ● An offshore banking unit may not enter into any transactions in foreign exchange with residents in India, unless such a person is eligible under the existing exchange control regulations to invest/maintain foreign currency accounts abroad. ● All prudential norms applicable to overseas branches of Indian banks apply to offshore banking units. The offshore banking units are also required to follow the best international practice of 90 days’ payment delinquency norm for income recognition, asset classification and provisioning. ● Offshore banking units are required to adopt liquidity and interest rate risk management policies prescribed by the RBI in respect of overseas branches of Indian banks as well as within the overall risk management and asset and liability management framework of the bank subject to monitoring by the bank’s board of directors at prescribed intervals. ● Offshore banking units may operate and maintain balance sheets only in foreign currency and are not allowed to deal in Indian rupees except for having a special rupee account out of the convertible funds in order to meet their daily expenses. These branches are prohibited from participating in the domestic call, notice, term etc. money market and payment system. ● The loans and advances of offshore banking units would not be reckoned as net bank credit for computing priority sector lending obligations. ● Offshore banking units must follow the ‘Know Your Customer’ guidelines and must be able to establish the identity and address of the participants in a transaction, the legal capacity of the participants and the identity of the beneficial owner of the funds. ● A bank cannot borrow from its offshore banking unit. ● The exposures of an offshore banking unit in the domestic tariff area should not exceed 25% of its total liabilities as at the close of business of the previous working day, at any point of time. Regulations and Guidelines of SEBI SEBI was established to protect the interests of public investors in securities and to promote the development of, and to regulate, the Indian securities market. We are subject to SEBI regulations for our equity and debt capital issuances, as well as our underwriting, banker to the issue, custodial, depositary participant, and debenture trusteeship activities. These regulations provide for our registration with the SEBI for each of these activities, functions and responsibilities. We are required to adhere to a code of conduct applicable for these activities. Foreign Ownership Restrictions Foreign investment in our Bank, as a corresponding new bank, is regulated by the provisions of the Bank Acquisition Act. Under Section 3(2D), foreign investment in new corresponding banks is subject to an overall statutory limit of 20% of the paid up capital. For public sector banks the RBI monitors the ceilings on Non Resident investments on a daily basis. For effective monitoring the RBI has fixed cut off points lower than the actual ceilings, which is 18% for public sector banks. Once the aggregate net purchases of equity shares of the respective bank by FIIs/NRIs/PIOs reaches the cut off points, which is 2% below the overall limit of 20%, the RBI cautions all designated bank branches so as not to purchase any more equity shares in the respective bank on behalf of NRIs/PIOs without prior approval of the RBI. The link offices are then required to intimate the RBI about the total number and value of equity shares/convertible debentures of the bank they propose to buy on behalf of FIIs/ NRIs/PIOs. On receipt of such proposals, the RBI gives clearances on a first-come first-serve basis till such investment in banks reach the applicable sectoral caps/statutory ceilings. On reaching the aggregate ceiling limit, the RBI advises all designated bank branches to stop purchases on behalf of their FIIs/NRIs/PIOs clients. 180 Special Status of Banks in India The special status of banks is recognised under various statutes including the Sick Industrial Companies Act, 1985, Recovery of Debts Due to Banks and Financial Institutions Act, 1993, and the Securitisation Act. As a bank, we are entitled to certain benefits under various statutes including the following: The Recovery of Debts Due to Banks and Financial Institutions Act, 1993 provides for establishment of Debt Recovery Tribunals for expeditious adjudication and recovery of debts due to any bank or Public Financial Institution or to a consortium of banks and Public Financial Institutions. Under this Act, the procedures for recoveries of debt have been simplified and time frames been fixed for speedy disposal of cases. Upon establishment of the Debt Recovery Tribunal, no court or other authority can exercise jurisdiction in relation to matters covered by this Act, except the higher courts in India in certain circumstances. The Sick Industrial Companies Act, 1985, provides for reference of sick industrial companies, to the Board for Industrial and Financial Reconstruction. Under it, other than the board of directors of a company, a scheduled bank (where it has an interest in the sick industrial company by any financial assistance or obligation, rendered by it or undertaken by it) may refer the company to the BIFR. The Sick Industrial Companies Act, 1985 has been repealed by the Sick Industrial Companies (Special Provisions) Repeal Act, 2004 (“SICA Repeal Act”). However, the SICA Repeal Act, which is due to come into force on a date to be notified by the Central Government in the official gazette, has not yet been notified. On the repeal becoming effective, the provisions of the Companies Act will apply in relation to sick companies, under which the reference must be made to the National Company Law Tribunal, and not the BIFR. The Securitisation Act focuses on improving the rights of banks and financial institutions and other specified secured creditors as well as asset reconstruction companies by providing that such secured creditors can take over management control of a borrower company upon default and/or sell assets without the intervention of courts, in accordance with the provisions of the Securitisation Act. Regulations Governing Insurance Companies Subsidiaries offering life insurance and non-life insurance are subject to the provisions of the Insurance Act, 1938 and the various regulations prescribed by the Insurance Regulatory and Development Authority. These regulations regulate and govern, among other things, registration as an insurance company, investment, licensing of insurance agents, advertising, sale and distribution of insurance products and services and protection of policyholders’ interests. In May 2002, the Indian parliament approved the Insurance (Amendment) Act 2002, which facilitates the appointment of corporate agents by insurance companies and prohibits intermediaries and brokers from operating as surrogate insurance agents. Regulations Governing International Businesses Our international business operations are governed by regulations of the countries where we have a presence. We are required to obtain approval of the RBI to set up overseas subsidiaries, offshore branches and representative offices abroad. Further, approval from the foreign regulatory authority is also required prior to undertaking such banking operations. Overseas Joint Venture We entered into a technical services (equity and management participation) agreement with the Everest Bank Limited, incorporated in Kathmandu under the laws of Nepal on July 3, 2001. Overseas branches We have established an overseas branch at Kabul, Afghanistan regulated by Da Afghanistan Bank. Representative offices We have representative offices at Almaty in Kazakhstan, Shanghai in China and London in the United Kingdom. 181 GOVERNMENT APPROVALS In view of the approvals listed below, we can undertake this Issue and our current business activities and no further major approvals from any Government authority/ RBI is required to continue these activities. A. Approvals for the Issue A.1. Government Approval: The Banking Division, Department of Economic Affairs, MoF, GoI has given permission for the Issue under Section 3(2B)(c) of the Bank Acquisition Act through letter number F.No.11/29/2004-BOA dated January 7, 2005. The contents of this approval are being reproduced and are as follows: “Dated the 7th January, 2005 To The Chairman & Managing Director Punjab National Bank Head Office New Delhi. Subject: Follow on Public Offer by Punjab National Bank of Rs. 80 crores Sir, I am directed to refer to Punjab National Bank’s letter No. Nil dated 14th September, 2004 on the above subject. The approval of the Central Government under Section 3 (2B) (c) of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 is hereby conveyed to Punjab National Bank for its follow-on public issue of Rs. 80 crores comprising eight crore equity shares of Rs. 10/- each at a premium to be decided by the bank subject to the following terms and conditions:- (i) The Bank should comply with the provisions of Section 3 of the Banking Companies (Acquisition & Transfer of Undertakings) Act, 1970; (ii) Central Government’s share holding shall not be below 51% at any point of time; (iii) The timing of the public issue shall be decided by the Bank keeping in view the conditions prevailing in the market; every effort may, however, be made to complete the process by 31st March, 2005. (iv) The Bank shall obtain necessary approvals from its Board of Directors/Management Committee of the Board of Directors, Securities & Exchange Board of India, and other Regulatory Bodies; (v) Reservation/firm allotment, if any, to financial institutions, banks, mutual funds, Non-Resident Indians/ Overseas Corporate Borrowers etc., should be advised to the Government of India/Reserve Bank of India before the issue is launched. Allotment to Non-Residents, if any, will be subject to prior approval of Exchange Control Department of Reserve Bank of India and should be within the ceiling of 20% of paid-up capital or any lower ceiling that may be notified by the Government of India under sub-Section (2D) of Section 3 of the Banking Companies (Acquisition & Transfer of Undertakings) Act, 1970. (vi) Out of the above eight crore shares the amount raised for three crore shares through public offer should be returned to Government of India at the issue price by 31st March 2005. (vii) The proportionate cost of public issue to the extent of three crore shares to be borne by the Government. (viii) The Bank should restrict its public issue expenses to the bare minimum. 2. As regards, grant of exemption from compliance of the provisions of Section 13 and Section 15(1) of the Banking Regulation Act, 1949, necessary Gazette notification in this regard will be issued separately. 3. The Bank may intimate to the Govt. the final issue structure, time of the offer etc. as soon as these are finalized. 182 4. The receipt of this letter may kindly be acknowledged. Yours Faithfully, (D.P. Bhardwaj) Under Secretary to the Government of India. Tel no. 23360357 Copy to the Chief General Manger, Department of Banking Operations and Development, RBI, Central Office, Mumbai with reference to their letter No. DBOD.BP.540/21.01.002/2004-05 dated 19th October, 2004” A.2. RBI Approval: We have received permission from the Foreign Exchange Department, RBI vide their letter no.FE.CO.FID/ 4998/10.02.40(8435)/2004-05 dated February 2, 2005 permitting us to issue shares to NRIs / FIIs with repatriation benefits out of our public Issue of 80,000,000 Equity Shares. The RBI has imposed the condition that post Issue the non resident equity holding in our Bank should not exceed 20% of the paid up capital. The permission is further subject to the conditions laid down by the Government of India in their approval letter no. F.No.11/29/2004-BOA dated January 7, 2005, condition prescribed/stipulated by SEBI in this connection and the terms and conditions for issue of shares as stipulated in the Schedule 1 and 2 to RBI Notification No. FEMA.20/2000-RB dated May 3, 2000. B. Approvals for Our Current Business Licences and Approvals from the Reserve Bank of India ● Section 22 of the Banking Regulation Act, which requires a licence to be obtained from the RBI in order to carry out banking business in India, applies only to banking companies, and not corresponding new banks. Accordingly, our Bank does not require a licence in order to carry out banking activities. ● Approval number DBOD No. IBS. 821/23.01.006/95-96 dated January 1, 1996 to establish a representative office at Kazakhstan. ● Approval number DBOD. IBS/ 1111/23.01.006/2003-04 dated January 17, 2004 to establish a branch at Kabul, Afghanistan. ● Approval number DBOD No. IBS. 1039/23.01.006/2002-03 dated December 2, 2002 to establish representative offices at Shanghai, China, Dubai, United Arab Emirates and London, UK. Approval valid for one year. Shanghai and London offices established under this approval. ● Approval number DBOD.IBS/240/23.01.006/2004-05 dated August 23, 2004 extending validity of approval number DBOD No. IBS. 1039/23.01.006/2002-03 dated December 2, 2002 for 6 months till December 1, 2004. ● Licence bearing number BL.M. 1345 vide letter number DBOD (MRO) No. BL 91/22/02/030/2003-04 dated July 29, 2003 to open offshore banking unit at Santacruz Electronic Exports Promotion Zone, Mumbai. ● Approval number DBOD No. IBS IISC/23.37.003/95-96 dated March 16, 1996 to participate in joint venture arrangement with Everest Bank Ltd., Nepal and to enter into a technical/ management service agreement with Everest Bank Ltd. ● The Bank has obtained from the RBI a licence to deal in foreign exchange. ● Licence bearing number BL.LK. 98/2003 vide letter number DBOD No. H.434/22.03.10/2003-2004 dated November 7, 2003 to open an offshore banking unit at the Noida Special Economic Zone. The approval is valid for one year, i.e., till November 6, 2004. ● Approval bearing number DBOD.FSC.No.765/24.11.003/2002-03 dated April 29, 2003 for entering into joint venture with Principal Financial Group of the USA for the purposes of setting up an asset management, trustee company and distribution company. ● In principle approval bearing number DBOD.No.FSC. /24.01.018/2002-03 dated April 21, 2003 to the Bank for acting as corporate agent to undertake distribution of life/non- life insurance products on agency basis without any risk participation. ● In principle approval bearing number DBOD.No.FSC.283/24.01.018/2003-04 for setting up a joint venture insurance broking company with Principal Financial Group, Berger Paints India Ltd., and Vijaya Bank. 183 Approvals Granted by the Insurance Regulatory and Development Authority ● No objection letter from the Insurance Regulatory and Development Authority dated July 9, 2004 bearing number IRDA/DB208/03 stating that the Insurance Regulatory and Development Authority has no objection to the Punjab National Bank and Vijaya Bank holding equity in a proposed joint venture. ● Licence dated May 7, 2003 bearing number 1174676 for working as a corporate agent for three years from May 7, 2003 for procuring or soliciting insurance business of one general insurer. Licences and Approvals from the Securities and Exchange Board of India ● Registration certificate dated December 1, 2003 under the SEBI (Bankers to an Issue) Regulations, 1994 for the period December 1, 2003 to November 30, 2006 permitting the bank to be a banker to an issue. The Bank’s registration code is INBI00000084. ● Registration certificate dated March 1, 2002 under the SEBI (Underwriters) Regulations, 1993 for the period March 1, 2002 to February 28, 2005 permitting the bank to be an underwriter. The Bank’s registration code is INU000000563. ● Registration certificate dated August 1, 2004 under the SEBI (Debenture Trustee) Regulations, 1993 for the period August 1, 2004 to July 31, 2007 permitting the bank to be a debenture trustee. The Bank’s registration code is IND000000023. ● Registration certificate dated October 6, 2003 under the SEBI (Depositories and Participants) Regulations, 1996 for the period October 6, 2003 to October 5, 2008 permitting the bank to be a Depository Participant. The Bank’s registration code is IN-DP-NSDL-68-98. Applications made by the Bank ● We have applied for extension of licence bearing number BL.LK. 98/2003 for opening an offshore banking unit at the Noida Special Economic Zone on October 8, 2004 to the RBI for extending the validity of the licence for a further period of one year. ● We have applied to the Governor of the Central Bank of the U.A.E. on August 7, 2003 requesting permission to establish a representative office at Dubai. ● We have applied to RBI vide letter bearing number BMS/INS/JV on August 1, 2003 for setting up a joint venture with Principal Financial Services Inc. for pension and life insurance company and insurance broking company with Principal Financial Services Inc. Licences and Approvals from foreign regulatory authorities ● Registration certificate dated August 14, 2003 from the Registrar of Companies for England and Wales for the establishment of a branch in England and Wales. The number assigned to the Bank is FCO24714 and to the branch is BR007210. ● Registration certificate number 25301 from Da Afghanistan Bank dated May 11, 2004 for the establishment of a banking branch to carry on the business of commercial banking in Afghanistan. ● Approval from the Supervising Management Committee of China Banking Regulatory Commission dated April 16, 2004 for the establishment of a representative office at Shanghai, China. ● Approval from the National Bank of the Republic of Kazakistan dated July 23, 1998 for the establishment of a representative office. ● Approval from the Nepal Rastra Bank dated August 6, 2001 vide letter B.R.D.2 (Everest) 5/058 approving renewal of the technical services agreement executed between the Bank and Everest Bank Limited. Approvals for Setting up Branch Offices We require prior approval from the RBI for opening a new place of business in India or abroad or for otherwise than within the same city, town or village, the location of the existing place of business. Except as stated above, we have obtained the necessary approvals from the appropriate statutory and regulatory authorities for carrying out our business and operations through our branches and representative offices and no further approvals are required from any government authority/ RBI to continue our business and operations. There are no approvals which have expired, or which have been applied for and have not been granted to the branches of the Bank. We do not require to apply for any other approvals for the purposes of running our business and operations. 184 OUTSTANDING LITIGATION AND MATERIAL DEVELOPMENTS Except as described below, there is no outstanding litigation, suits or criminal or civil prosecutions, proceedings or tax liabilities against our Bank, its Directors, our subsidiaries, joint ventures and regional rural banks sponsored by our Bank, that would have a material adverse effect on our business and there are no defaults, non-payment or overdue of statutory dues, institutional/ bank dues and dues payable to holders of any debentures, bonds and fixed deposits that would have a material adverse effect on our business other than unclaimed liabilities against our Bank or Directors, our subsidiaries, joint ventures and regional rural banks sponsored by our Bank. Contingent liabilities not provided for as of March 31, 2004: Rs. 322,300,265 (as compared to Rs. 235,708,644 as of March 31, 2003). I. Litigation against our Bank A. Pending Litigation Against Our Bank 1. Criminal Cases (i) There are two criminal cases pending against our Bank. The details of these cases are as follows: (a) The Department of Banking Operations and Development of the RBI in Trivandrum, Kerala has filed a case against Nedungadi Bank (which has now been merged with our Bank) and the former chairman of Nedungadi Bank, Mr. A.R. Moorthy. The complainant has filed the case, C.R.P 15 of 2002 in the court of the Judicial First Class Magistrate, Kozhikode under Section 46 of the Banking Regulation Act and Section 190 (1) of the Code of Criminal Procedure, 1973. The complainant has alleged that in the course of the annual financial inspection by the officials of the RBI, the inspecting officials found that there were several irregularities of serious nature in the bank’s lending and investment operations with reference to the financial position as of December 31, 2000. The complainant has drawn notice to the following directions of the RBI: (a) that investments in and underwriting of shares and debentures of corporate bodies have been permitted only up to 50% of the incremental deposits of the previous year and (b) that the bank should not grant loans, advances, etc to relatives of the chairman, managing director or directors of the bank above Rs. 2.5 million without the approval of the board of directors. The complainant has alleged that Nedungadi Bank has approved a scheme in September 1999 envisaging the sale and purchase of shares in order to take advantage of price difference between the BSE and NSE and other stock exchanges. The Nedungadi Bank allegedly engaged three broking firms for this purpose and as of March 31, 2000, an amount of Rs. 945.2 million had become receivable by Nedungadi Bank, which was shown as ‘other assets’ in the balance sheets. The RBI has clarified the scope of the entry of ‘other assets’. The complainant has alleged that the receivables amounting to Rs. 945.2 million have been wrongly and incorrectly shown in the balance sheet of Nedungadi Bank. The complainant has also alleged that loans, advances above Rs. 2.5 million have been made to relatives of the chairman, managing director or directors of the bank without the approval of the board of directors of Nedungadi Bank. The complaint is dated May 14, 2001, which was prior to the merger of Nedungadi Bank with our Bank. However, as Nedungadi Bank has merged with our Bank, we have been made a party to the case. The case has not come up for hearing and evidence has not been adduced. (b) UTI Bank has filed a First Information Report (FIR) with Navrangpura Police Station against our Bank in relation to an alleged fraudulent encashment of cheques for an amount of approximately Rs. 95,000 by Nedungadi Bank. The FIR was filed prior to the merger of Nedungadi Bank with our Bank. However, as Nedungadi Bank has merged with our Bank, we have been made a party to the case. The police is yet to conduct an enquiry. (ii) There is one criminal complaint against Mr. S.S. Kohli, the Chairman and Managing Director of our Bank. The details of this case is as follows: Mr. M.M. Sehgal has filed a criminal complaint against Mr. S.S. Kohli, Chairman and Managing Director of our Bank among 36 other respondents. Mr. S.S. Kohli has been named as the 13th accused in this complaint. The complaint has been filed in the court of Chief Metropolitan Magistrate as criminal complaint no. 64 of 2002. The complainant is the former chairman of a company (Sehgal Papers Limited), which is under liquidation. The complainant has alleged that the accused have entered into a criminal conspiracy and have acted in a manner so as to bring the complainants company into ruinous state by the abuse of their powers. He has alleged that the support of the banks, including our Bank was 185 essential for the success of his company. It has been alleged that the Bank had promised assistance by way of loan amounting to Rs.7.9 million to Mr. Sehgal’s company. He has alleged that the Bank has not provided such support and has therefore filed the said criminal complaint. Notice has not yet been served by the court on the accused in this case. (iii) A writ petition, WP 6412 of 2004 has been filed by an association of the shareholders of Nedungadi Bank in the High Court of Kerala against our Bank and some employees. The petitioners have prayed for a writ of mandamus to have an enquiry into the alleged grave manipulation of money running into several crores of rupees and to punish these employees under the penal laws of India. 2. Cases related to statutory authorities. A. Income Tax Cases There are disputes relating to income tax assessments of the assessment years 1992-1993, 1994-1995 to 2003- 2004. The total amount claimed against our Bank in relation to income tax as of December 31, 2004 including interest is approximately Rs. 485.94 million and our Bank has already paid the entire amount claimed by the Income Tax Department (“IT Department”) under protest except Rs. 71 million in relation to assessment year 1997-98 on account of disallowance of software and bond issue expenditure due to rectification application filed and pending with the IT Department. There are three main issues, which are points of disputes between the IT Department and our Bank. The total liability of our Bank with regard to these main issues is Rs. 267.52 million. The remaining Rs. 218.42 million is on account of disputes on matters such as guesthouse expenses, holiday home expenses, depreciation on let out buildings, interest tax, etc. (a) Disallowance of exempted income from interest on loans to infrastructure projects: Our Bank extends loans to entities involved in infrastructure projects. The income of our Bank from such loans is exempted from computation of total income under Section 10(23G) of the I.T. Act. The exemption is allowed under Section 10(23G) of the I.T. Act only for such infrastructure projects, which have been approved by the central government. Our Bank has claimed exemption from computation of total income for incomes from some infrastructure projects, which have not yet received the approval from the central government. The total liability of our Bank in this regard amounts to Rs.191.27 million. The Commissioner of Income Tax (Appeals), New Delhi (“CIT”) has directed the assessing officer vide order dated July 29, 2004 and October 5, 2004 to consider the claim of our Bank in case the approvals from the central government are issued with retrospective effect and pass necessary rectification orders to allow the claims of our Bank on production of necessary approvals. The Bank is awaiting approvals of the Central Board of Direct Taxes for confirming the respective projects as notified projects. In the event a project is so notified, the Bank’s claim will be realised. (b) Disallowance of depreciation on investment in securities: Our Bank has invested in securities. Our Bank has claimed that under the guidelines of the RBI, the value of the securities is accounted as stock in trade and on the cost price of the securities and the market price of the securities, whichever is lower. In the event the market price is lesser than the cost price of the securities, the difference is treated as depreciation by our Bank in accordance with the applicable RBI guidelines. Hence, treatment of the investments in securities as depreciation results in lowering of the taxable income of our Bank as depreciation of securities is treated as expenditure. The Supreme Court of India has held that such treatment of depreciation as expenditure is allowable in the case of UCO Bank v. Union of India (24 ITR 355). The IT Department has claimed that the loss is notional in nature and therefore has disallowed the claim of depreciation by our Bank. The issue is pending in Income Tax Appellate Tribunal (“ITAT”) and pending the decision of the ITAT, the IT Department has continued to impose liability on our Bank. The total amount of claims against our Bank in this regard amounts to Rs.14.80 million. (c) Disallowance of provisions for bad and doubtful debts as deductions from taxable income: Our Bank has made provisions for bad and doubtful debts in its books. The provisions of Section 36(1)(viia) of the I.T. Act, allow deduction in computing the income in respect of any provision for bad and doubtful debts made by a scheduled bank upto an amount which is the aggregate of a certain percentage of the total 186 bank and the average advances made by rural branches of such bank. Our Bank has claimed deduction for provisions for bad and doubtful debts as deduction from income. The assessing officer has disagreed with the extent of the deduction, which can be claimed by our Bank. The total liability of our Bank in this regard amounts to Rs.61.45 million. The income tax related cases in the various financial years are as follows: (i) For assessment year 1992-1993: The IT Department has imposed a liability of Rs.0.34 million on our Bank. The liability has been imposed on account of alleged deficiencies in certificates evidencing payment of tax deductible at source. Our Bank has filed a rectification application with the assessing officer. The matter is pending before the assessing officer. (ii) For assessment year 1994-1995: The IT Department has imposed a liability of Rs.39.45 million on our Bank. The liability has been imposed on account of loss claimed by our Bank on certain bonds subscribed to by our Bank and disallowances on interest tax. The matter is pending in the ITAT. (iii) For assessment year 1995- 1996: The IT Department has imposed a liability of Rs.2.51 million on our Bank. The liability has been imposed on account of loss claimed by our Bank on certain bonds subscribed to by our Bank and disallowances on interest tax. The matter is pending in the ITAT. (iv) For assessment year 1996- 1997: The IT Department has imposed a liability of Rs.91.67 million on our Bank. The liability has been imposed on account of disallowance of provisions for bad and doubtful debts as deductions, disallowance of depreciation on investment in securities, disallowance of holiday home expenditure, etc from taxable income. The matters are pending in the ITAT and before the CIT. (v) For assessment year 1997-1998: The IT Department has imposed a liability of Rs.83.04 million on our Bank. The liability has been imposed on account of disallowance of provisions for bad and doubtful debts as deductions, disallowance of depreciation on investment in securities from the income, additions by the assessing officer to the income on account of certain recoveries, which had been treated previously as bad and doubtful debts and which are refunded to the insurer. The matters are pending in the ITAT. (vi) For assessment year 1998-1999: The IT Department has imposed a liability of Rs. 24.81 million on our Bank. The liability has been imposed on account of addition of income generated through interest on tax free securities by the assessing officer, addition of income from interest on non performing assets by the assessing officer and disallowance of provisions for bad and doubtful debts as deductions. The matter is pending in the ITAT. (vii) For assessment year 1999-2000: The IT Department has imposed a liability of Rs. 26.67 million on our Bank. The liability has been imposed on account of disallowances of expenditure of software and bond issue by the assessing officer. The matter is pending before the Commissioner of Income Tax (Appeals). (viii) For assessment year 2000-2001: The IT Department has imposed a liability of Rs. 24.86 million on our Bank. The liability has been imposed on account of disallowances of expenditure of software and bond issue by the assessing officer. The matter is pending before the Commissioner of Income Tax (Appeals). (ix) For assessment year 2001-2002: The IT Department has imposed a liability of Rs. 1.32 million on our Bank. The liability has been imposed on account of alleged deficiencies in certificates evidencing payment of tax deductible at 187 source. Our Bank has filed a rectification application with the assessing officer. The assessing officer rejected the application. Our Bank has filed an appeal in the ITAT. The matter is pending in the ITAT. (viii) For assessment year 2002-2003: The IT Department has imposed a liability of Rs. 112 million on our Bank. The liability has been imposed on account of disallowance of exempted income from interest on loans to infrastructure projects. (ix) For assessment year 2003-2004: The IT Department has imposed a liability of Rs. 79.27 million on our Bank. The liability has been imposed on account of disallowance of exempted income from interest on loans to infrastructure projects. B. Sales Tax case The Commercial Sales Tax Department of the Government of Rajasthan has served notice to the Bank for non payment of tax payable for dealing in bullion, vide order dated September 12, 2003. The Commercial Sales Tax Department has imposed a liability of Rs. 66,65,494 on the Bank. However, a sum of Rs. 20,81,341 has already been paid as per order of the tax department and therefore, the aforesaid demand is reduced to Rs. 45, 84,153. Our Bank has appealed against the said order and has filed its appeal before the Deputy Commissioner (Appeals) Zone-II, Commercial Tax Department, Jaipur. C. Cases relating to Foreign Exchange Regulations There are two cases against our Bank relating to alleged violations of foreign exchange regulations in India. The total amount claimed against the Bank is Rs.3.5 million. The details of these two cases are as follows: (i) The Director (Special) Enforcement has imposed a fine of Rs.2.0 million through order dated August 5, 2004 and alleged that in the year 1993-1994, our Bank permitted certain deposits by a holder of a NRE account even though the status of this holder as a NRE had become doubtful and therefore, the Bank was negligent and was liable to pay penalty. Our Bank filed appeal before the appellate tribunal under FEMA. The appellate tribunal has sought the furnishing of a bank guarantee amounting to Rs.2.0 million, which the Bank has furnished on December 4, 2004. (ii) The Director (Special) Enforcement has imposed a fine of Rs.1.5 million on our Bank through an order dated October 29, 2004. The Director (Special) Enforcement has alleged that our Bank had acted negligently by acting as the authorised branch through which the exporter sought reimbursement from the RBI, even though the goods never reached their intended destination, Russia and were instead unloaded in Dubai. The Bank appealed before the appellate tribunal under FEMA. The matter is pending before the appellate tribunal. 3. Cases pertaining to fraudulent transactions There are 79 cases filed against our Bank for disputes in relation to forged signatures, non-tallying of signatures, breach of trust and fraudulent withdrawal of money by allegedly unauthorised persons including our employees. The total amount claimed by the petitioners in these cases amounts to Rs. 313,850,933 . There are three material cases in this regard. The details of the same are as follows: (i) Roop Polymers Limited has filed a case, Roop Polymers Ltd. v. Punjab National Bank, case number 62/04, against eleven parties including our Bank for recovery of Rs. 26,981,661 before the Civil Court, Gurgaon, on account of alleged fraudulent withdrawals from its account by its employee. The plaintiff claims that the documents related to electronic transfer show that the signatures of the plaintiff’s directors were forged and the signatures do not remotely resemble the specimen signatures of the authorized signatories of the plaintiff, which are in the records of the Bank. Hence, the plaintiff has made a claim for damages against our Bank for acting recklessly and negligently while making payments against forged cheques and for making unauthorized electronic transfers. The matter is pending in the Civil Court, Gurgaon. (ii) Allahabad Bank has filed a suit, Allahabad Bank v. Punjab National Bank, suit no. 7/99 against our Bank in the District Court, Alipore for recovery of Rs. 255,526,698. The plaintiff has contended that it had issued a bankers cheque without any consideration and the same was wrongfully encashed in the accounts of various parties by our Bank. The case is listed for completion of service of summons. 188 (iii) Ceat Tyres Limited has filed suit, Ceat Tyres Limited v. Punjab National Bank and others, suit no. 128/1997 in the Mumbai High Court for recovery of Rs. 17,373,949 alleging that our Bank collected certain forged cheques, acted negligently and was therefore instrumental to the commission of fraud. Our Bank has filed a written statement and the matter is pending hearing in the Mumbai High Court. 4. Cases pertaining to facilities provided by our Bank There are 104 cases filed against our Bank relating to disputes on account of alleged non-deposit, non-realisation or loss of cheques and alleged wrongful refusal to sanction certain facilities/overdrafts by our Bank or release of sums to certain persons or wrongful debits in a customer’s account or refusal to honour bank guarantees, letters of credits, bills of lading and other such instruments. Some of these cases also relate to the fixed deposit receipts maintained at our Bank by our customers. The total amount claimed in the aforementioned cases is Rs. 19,331,150,550 . There are eight material cases in this regard. The details of the same are as follows: (i) Trihut Tannery Private Limited has alleged in Trihut Tannery Pvt. Ltd v. PNB Branch Office (Jawaharlal Road Muzzarffarpur), suit no. PT 212/9 before the Civil Court, Muzzarffarpur claiming that as a result of under-financing by our Bank, it could not make payment of dues to its creditors and has therefore, suffered loss of profit, loss of business and loss of reputation. The plaintiff has claimed a sum of Rs. 22,576,445 as damages and compensation for pecuniary loss allegedly caused by our Bank. An application for transfer of the case to Debt Recovery Tribunal, Patna has been filed before the Civil Court, Muzzaffarpur and the same is pending. (ii) Abhishek Pharmaceuticals has filed a suit, Abhishek Pharmaceutical v. Punjab National Bank and others, suit no. 23/1994, against our Bank before the Court of Subordinate Judge, Jehanabad for alleged wrongful refusal by our Bank to enhance the working capital limits previously sanctioned to the plaintiff. The plaintiff has claimed that our Bank acted negligently and did not enhance the sanctioned credit limits. The plaintiff has claimed an amount of Rs. 20,100,000. The case is pending before Court of Subordinate Judge, Jehanabad. (iii) Savik Vijay Engineering Private Limited has filed a suit, Savik Vijay Engineering Private Limited v. Punjab National Bank, suit no 1448/98 in the City Civil Court, Bangalore and claimed that our Bank wilfully delayed the release of the loan and therefore, the factory of the plaintiff could not be rehabilitated and production could not be started. The plaintiff has filed a recovery suit for damages of Rs. 50,000,000 and for a direction to release the sanctioned loan of Rs. 20,000,000. The Bank has filed its reply and the case is pending at the stage of evidence. (iv) Modi Rubber Limited has filed a case, Modi Rubber Limited v. Punjab National Bank, suit no 2761/95 in the Delhi High Court against our Bank for alleged arbitrary appropriation of its fixed deposit receipts worth Rs. 34,961,000 by the Bank towards the bonus term loan account of Modi Spinning and Weaving Mills Limited since the plaintiff was the guarantor for Modi Spinning and Weaving Mills Limited. The amount claimed by the plaintiff is Rs. 54,367,606. The Delhi High Court has sine die adjourned this case in light of the fact that the proceedings for Modi Spinning and Weaving Mills Limited are pending before the BIFR. (v) ATV Projects India Limited has filed a suit ATV Projects India Limited v. Punjab National Bank bearing number 198/ 2001 in the Mumbai High Court claiming damages against, inter alia, our Bank, alleging that it incurred a total loss of Rs. 3,059,600,000 which has been caused due to delay in the sanctioning and disbursement of working capital to the plaintiff. The claim against our Bank is Rs. 284,800,000. The matter is pending in the Mumbai High Court. (vi) Ruby Tea Estate has claimed Rs. 43,000,000 in a suit, Ruby Tea Estate v. Punjab National Bank, suit no. 280/96 before the District Court, Alipore. The plaintiff has alleged that our Bank refused to enhance the credit limit of the plaintiff and the same resulted in significant loss to the plaintiff. The matter is pending hearing and final adjudication in the District Court, Alipore. (vi) K.R. Steel Union Limited has filed a suit, K.R. Steel Union Limited v. Punjab National Bank, no. 1933/98 wherein the plaintiff has claimed that our Bank failed to open letters of credit which had been allegedly sanctioned by our Bank and has claimed damages of Rs. 528,479,999on account of loss of business opportunities and compensation for hardship. The suit is pending for hearing in the Mumbai High Court. (vii) B.S.M. Knit Fab (India) Limited has filed an application to sue as an indigent person in the Court of Civil Judge, Junior Division, Chandigarh against our Bank, Industrial Development Bank of India and Punjab State Industrial Development Corporation, claiming damages of Rs. 18,252,400,000 alongwith interest at the rate of 15% on 189 account of alleged delay in opening of letters of credit and re-scheduling of loans etc. The case is fixed for hearing on March 1, 2005. (viii) Our Bank and Topline Shoes Limited has filed appeals before the Debt Recovery Appellate Tribunal, Mumbai in the case of Topline Shoes Limited v. Punjab National Bank (W.P. 9842/2004). The plaintiff had opened a current account for payment of refunded orders but certain cheques issued by the plaintiff were dishonoured. A sum of Rs. 17,754,990 has been claimed against our Bank. The case is pending hearing and final adjudication. 5. Consumer Cases There are 211 cases filed against our Bank in district, state and national consumer forum alleging, among other things, deficiencies in our services. The total amount claimed in such cases is Rs. 78,854,216. There is one material case in this regard. The details of the same are as follows: NSIC Limited has filed a case against us in the National Consumer Dispute Redressal Forum, New Delhi. NSIC Limited has claimed an amount of Rs 21,281,971 against our Bank on account of certain letters of credit sent for collection by us and which were lost in transit. 6. Cases pertaining to interest charges There are six cases pending against our Bank wherein disputes have arisen in relation to the interest charged or leviable by our Bank. The cumulative amount claimed in these cases is Rs. 7,491,425 . 7. Counter claims in recovery suits filed by our Bank In the event a customer fails to pay us the amounts due to us within the prescribed time, we take necessary actions to recover such amounts. Such actions include enforcement of the security or filing of recovery suits against the defaulting customers. A number of such customers have filed counter claims and counter suits against recovery suits or other actions undertaken by our Bank. There are 24 such cases pending against our Bank and the total amount in dispute is Rs.230,706,957. There are five material cases in this regard. The details of the same are as follows: (i) We had filed a suit against R K Solvent for recovery of dues payable to us. However, the R K Solvent has filed a suit, R K Solvent v. PNB Branch Office (Boring Road, Patna) and others, against our Bank for recovery of Rs. 22,091,977 on account of loss of expected profits and interests due to alleged vindictive attitude of our Bank, which involved filing of criminal cases against the directors of R K Solvent. R K Solvent has also claimed that our Bank did not act in a timely manner to get the consignment released which resulted in pecuniary loss and loss of goodwill to R K Solvent. The case has been transferred to the DRT, Patna. (ii) Our Bank had filed recovery suits against the various companies of the SRC Group. SRC Group has filed 11 counter claims in these suits (five of these cases are above Rs. 10,000,000). The Debt Recovery Tribunal, Chennai had dismissed their application on account on non-payment of court fee. The plaintiffs moved an application for restoration and the matter is currently pending in DRT, Chennai. The total amount counterclaimed against our Bank is Rs.47,500,000. (iii) DIU Steels filed a civil suit, DIU Steels v. Punjab National Bank in the Kolkata High Court bearing number C.S. 133 of 2001 against us for recovery of Rs.100,000,000. The suit has been filed against our Bank wherein the plaintiff alleges that our Bank has taken over the hypothecated stocks and other assets of the plaintiff, without reasonable notice. Our Bank has filed its written statement and the matter is pending for hearing in the Kolkata High Court. (iv) Surgiplast Limited has filed a civil suit no. 556/93 in the City Civil Court at Ahmedabad against our Bank for recovery of Rs.16,384,000. The plaintiff has alleged that our Bank converted the hypothecated goods into pledge and as a result, the hypothecated goods lost their validity period. Therefore, the plaintiffs have claimed that it suffered losses to the extent of Rs.16,384,000. Our Bank has filed a written statement and the matter is pending hearing and final adjudication in the City Civil Court at Ahmedabad. (v) Shree Swaminarayan and company has filed a suit no. 3991/90 against our Bank in the City Civil Court, Ahmedabad against our Bank for recovery of Rs.11,875,520 and also a declaration that mortgaged documents cannot be enforced against the borrower and its sureties. The plaintiff has further alleged that our Bank wrongfully refused to allow the plaintiff to withdraw the pledged goods, which were essential for charging its plant and machinery. The suit has been transferred to the DRT for final disposal. 190 8. Property disputes A number of our branches are located on rented premises. As a result, 17 cases have been filed against our Bank in relation to arrears/enhancement of the rent of the present/earlier premises occupied by our Bank. The total amount claimed in such cases is Rs. 53,496,631 . There are two material cases in this regard and the details of the same are as follows: (i) Mr. Bhagat Ram Arora v. PNB – The landlord of the premises of one of our branches in Kolkata has filed an eviction suit number 591/99 against us at the Kolkata High Court wherein he has claimed damages amounting to Rs.32,290,956 for wrongful use of the premises. The Bank has filed the written statement and the matter is pending for hearing. (ii) Chunnilal and Company v. PNB - The plaintiff had rented the premises to one of our branches in Mumbai as a ‘sub- tenant’ and has filed an eviction suit number 167/176 of 2002 in the Small Causes Court wherein he claimed rent for Rs. 3,63,654 per month and a total amount of approximately Rs. 10,379,319. The Bank has filed a written statement and the matter is pending for hearing. 9. Cases wherein our Bank has been impleaded as a third party Our Bank is often impleaded as a third party in a dispute between two persons wherein one of the persons may be a customer of our Bank and therefore, the court may order the parties to recover the sums due from the assets of such customer at our Bank. There are 12 such cases currently pending at various courts in India and the total amount claimed in such cases is Rs. 28,669,542. There are two material cases in this regard. The details of the same are as follows: (i) Uniscan Sonics Limited had allegedly drawn various bills in favour of United HealthCare Limited. Uniscan Sonics Limited has also alleged that our Bank has wrongfully co-accepted the same. The plaintiffs failed to honour the bills and therefore, our Bank was asked to pay an amount of Rs.26,710,865 to Canara Bank . However, our Bank has refused to honour the bills in light of Section 22 of the Negotiable Instruments Act, 1938. Hence, the plaintiff has filed a case, Uniscan Sonics Limited v. Punjab National bank, suit no. 1260/91 in the District Court. The case has been transferred to the Debt Recovery Tribunal, Mumbai. The DRT has passed a stay order in this case. (ii) A suit, NSIC Limited v. Moti Industries Limited and Punjab National Bank, suit no. 3709/99 has been filed by NSIC Limited against Moti Industries Limited and our Bank in the Mumbai High Court, for a decree of Rs.23,592,259. This is the value of two letters of credit opened by our Bank in case the said amount is not paid by Moti Industries Limited to the plaintiff. The matter is pending hearing and final adjudication in the Mumbai High Court. 10. Labour related cases There are 701 labour related cases pending against our Bank in the labour courts and various high courts in India. The total amount claimed by the plaintiffs in these cases is approximately Rs.161.77 million. The aforesaid cases are classified as follows: (i) There are 263 cases pending against our Bank wherein employees have claimed payment of arrears, which includes payment of various allowances and reimbursements, gratuity, pension, differences on account of revised pay scales and other dues allegedly denied by us. The total claim arising out of these cases is approximately Rs. 17. 75 million (ii) There are 183 cases against our Bank for alleged wrongful dismissals and removal from service by us wherein the total amount claimed as compensation is approximately Rs. 90.63 million. (iii) There are 181 cases pending against our Bank for matters pertaining to issues such as promotion, reduction of grade/post, compulsory retirement, absorption, regularization, reinstatement, suspension, etc of our employees. The total claim against our Bank in these cases amounts to approximately Rs. 23.57 million. 191 (iv) There are 75 cases filed against our Bank for dues claimed by employees in relation to our voluntary retirement scheme. The total amount of such claims is approximately Rs. 29.81 million. There are two material cases that involve trade unions of our Bank. The details of these cases are as follows: (a) Mr. S.M. Goel and Punjab National Bank Retired Officers Union, the petitioners, S.M. Goel and Punjab National Bank Retired Officers Union v. Punjab National Bank, C.W.P. No. 5284/93 before the Delhi High Court, have alleged that our Bank unlawfully started recovering the ad-interim bonus paid by itself in 1974 from the salaries of the officers. This alleged action of our Bank has been challenged in the writ petition. The matter has been admitted by the Delhi High Court and is pending hearing and final adjudication. (b) A writ petition, Hindustan Commercial Bank Limited v. All India Hindustan Commercial Bank Employee Association and others, no. 8633186 was filed by Hindustan Commercial Bank Limited (HCB), which was merged with our Bank, in the Allahabad High Court against an award passed by CGIT, Kanpur, whereby HCB was ordered to allow the benefits of past temporary service to certain workmen who were given confirmed employment pursuant to a settlement dated April 25, 1978 entered into between the employees’ union of HCB and HCB. The operation of the said award was stayed by the Allahabad High Court. Our Bank has filed an application for impleading in the said writ petition and the same has been accepted by the Allahabad High Court. The Writ Petition will now come up for hearing in due course. 11. There are 13 notices against some branches of our Bank in which irregularities with regard to the public issues of certain other companies in which our Bank was acting as a intermediary and in which action has been taken by regulators against the said branches of the Bank. The details of the same are as follows: S. No. Branch of the Bank Name of issuer Irregularities Action Taken/Development company 1 Navrangpura, M/s Kangold (India) Branch accepted Branch was de-authorised for any type of Ahmedabad Ltd. application money after merchant banking business vide circular the closure of the issue dated June 11, 1996. The branch is still during October, 1994 de-authorised 2. Navrangpura, M/s Elvis India Ltd. Branch accepted 40 Bank dismissed from the bank’s service Ahmedabad applications with stock the then manager. Another officer was invests after closure of penalized by reducing his scale of pay by the issue in January, four stages. SEBI imposed a penalty of 1995 warning to the bank to be more careful in future vide its letter dated September 3, 2002. 3. M I Road, Jaipur M/s Asian Industrial Branch accepted 24 Branch was de-authorised for any type of Structures Ltd. applications in stock merchant banking business for one year invests amounting to Rs. vide circular dated June 23, 1997. 49.8 million after closure However, branch was again authorized to of the issue in January, handle merchant banking business vide 1996 circular dated July 25, 2000 as per recommendations of the zonal manager, Jaipur. Bank imposed upon the erring official of the branch penalty of dismissal from service. 192 S. No. Branch of the Bank Name of issuer Irregularities Action Taken/Development company 4. New Cloth Market, M/s Hindustan Fin. 19 applications In terms of hearing dated August 10, Ahmedabad Stock Ltd. amounting to Rs. 15.1 1999, SEBI recommended suspension of million were received registration of the branch for a period of 3 after closure of the months. Branch requested SEBI to take a public issue. lenient view in the light of the remedial measures taken by the branch. Branch is still deauthorised for accepting merchant banking business. 5. Ashram Road, M/s Renco Gears Application money was Branch was deauthorised for doing all Ahmedabad Ltd. released to the types of merchant banking business till company even before August 4, 2000. Disciplinary authority stock exchange had imposed a minor penalty on the then approval had been chief manager of the branch. obtained in 1995. 6. Relief Road, M/s Dhanlaxmi Branch accepted Non-maintenance of the record was Ahmedabad Lease Finance Ltd. applications after closing admitted by the bank. SEBI vide letter date of issue and failed dated October 19, 2002 imposed a penalty to maintain proper of debar on the branch for 6 months to records pertaining to the undertake any banker to issue business. issue in 1995. 7. Mahajan Gali, Vadodra M/s Rich Paints Ltd. Branch issued 6 stock Branch was deauthorised for doing all invests of Rs. 56 million types of merchant banking business till each aggregating Rs. 30 August 4, 2000. Disciplinary authority had million in the names of imposed a minor penalty on the then chief various persons in manager of the branch. violation of guidelines in 1996. 8. Raopura, Vadodra M/s Incap Financial Application of M/s Branch was deauthorised for acting as Services Ltd. Darshan Investments banker to an issue for 3 months vide Pvt. Ltd. for 600,000 circular dated March 27, 2000. Note of shares amounting to Rs. caution had been served to the erring 60 million was accepted official. and released after the closure of the public issue in early nineties. 9. Shahibaug, M/s Maha Application collected Staff side action was initiated against two Ahmedabad Chemicals Ltd. by the branch included officers and minor penalty of ‘censure’ 23 applications was imposed upon them by the Bank. 2 accompanied by stock months de-authorisation of the branch invests issued by Tamil was imposed by SEBI vide order dated Mercantile Bank, Unjha September 10, 2003 effective for 3 branch and these were weeks from the date of the order. 193 S. No. Branch of the Bank Name of issuer Irregularities Action Taken/Development company issued in the last week of May, 1994 and in the first week of June, 1994 whereas public issue of the company closed on April 15, 1994. 10. Navrangpura, M/s Saket Extrusion Stock invests of Rs. 15 SEBI on February 20, 2000 had recorded Ahmedabad million had 4 different statement of the branch manager. Staff dates as February 22, side was decided and the then manager 1994, March 30, 1994, was dismissed. Another officer was April 26, 1994 and penalized by reducing 4 stages in the March 27, 1994 in the scale. RBI was informed on May 9, 2001 columns meant for date to close the file. of issue whereas their presentation date was March 10, 1994. These were cancelled on May 26, 1994 despite various irregularities. 11. Sector 17-B, M/s Majestic Issue had not been fully Delay in clearance was due to rush of Chandigarh Industries subscribed on the day of closing work at the controlling branch. In closure i.e., April 10, personal hearing dated September 6, 1996. There was a delay 2004, SEBI was informed that there were of 10-12 days in holidays on account of Ambedkar Jayanti, clearance of cheques Ram Navmi etc. which also caused the enclosed with delay. Staff side is decided. SEBI has been application. requested to close the file. SEBI issued show cause notice on December 15, 2004 in relation to levy of minor penalty of suspension of the branch for undertaking bankers to issue transactions for a period of one month. 12. Erstwhile PNB Capital M/s Mefcom PNB Caps as lead SEBI proposed warning to erstwhile PNB Services. Markets Ltd. manager did not Caps for the violation. SEBI has been independently verify requested to take lenient view as PNB the claims of the Caps acted diligently and in compliance company for charging with the then existing guidelines of SEBI. Rs. 60/- per share for a Moreover, PNB Capital Services stands share of Rs. 10/- but merged with PNB. instead relied upon the statement of the company. 194 S. No. Branch of the Bank Name of issuer Irregularities Action Taken/Development company 13. Navrangpura, M/s Growmore Branch not only issued SEBI was informed on January 8, 2004 Ahmedabad Solvent Ltd. stock invests but also that Bank has already taken appropriate accepted applications action against the officer. SEBI vide letter subscribing to initial dated March 12, 2004 has recommended public offer & these a penalty of debarring the branch from were also used for carrying out activities of banker to an another company Saket issue for a period of one month. The Extrusions Ltd. branch is already deauthorised by the bank conduct merchant banking business. 12. Miscellaneous cases There are 32 other cases pending against our Bank, which have arisen either in connection with the death of the account holder or under the provisions of the Monopolies and Restrictive Trade Practices Act, 1969 or as a result of consolidation or shifting of our branches. The total amount claimed in such cases is approximately Rs.26.05 million. II. Litigation against Our Directors Except as disclosed below, there is no outstanding litigations against the directors of our Bank towards tax liabilities, criminal/civil prosecution for any offences (irrespective of whether they are offences in the nature of those specified under paragraph (i) of Part 1 of Schedule XIII of the Companies Act), disputes, defaults, non payment of statutory dues, proceedings initiated for economic offences, in their individual capacity or in connection with our Bank and other companies with which the Directors are associated: (i) There is one criminal complaint against Mr. S.S. Kohli, the Chairman and Managing Director of our Bank. For details of the case please see the details of criminal cases. (ii) A complaint has been filed under Indian Penal Code against M/s. Delfin Finance Ltd, in which Mr. Mohanjit Singh, one of our Directors is director, regarding pre-emptive purchase of property under provisions of the IT Act. III. Material Developments (a) Our Board of Directors has accorded in principle approval to a merger between IFCI and our bank, subject to certain terms and conditions. See the section titled “Risk Factors” on page x. (b) Our Board of Directors had authorised a fresh issue of up to 50,000,000 Equity Shares pursuant to a resolution passed at its meeting held on September 11, 2004. Our shareholders subsequently authorised the fresh issue of up to 50,000,000 Equity Shares, by a resolution passed unanimously at the general meeting of our Bank held on October 11, 2004, subject to the approval of the Government, the RBI, SEBI and other applicable authorities. The shareholder resolution gave the Board the authority and power to accept any modification in the proposal as may be required or imposed by such authorities and as agreed to by the Board. Our Bank applied to the Government on October 14, 2004 for its consent to a fresh issue of up to 50,000,000 Equity Shares. By its letter bearing number F.No.11/29/2004-BOA dated January 7, 2005, the Department of Economic Affairs, MoF, GoI granted its consent to the fresh issue of 80,000,000 Equity Shares, pursuant to Section 3 (2B) (c) of the Bank Acquisition Act. The Government Approval contained, inter alia, a condition that out of the 80,000,000 Equity Shares, the amount raised for 30,000,000 Equity Shares through the public offer should be returned to the Government at the Issue Price by March 31, 2005. The shareholders of the Bank, at an annual general meeting held on August 4, 2003, had approved a return of capital to the GoI to the extent of 130,000,000 Equity Shares or such other number of Equity Shares in one or more tranches and authorized the Board to accept, agree or otherwise to any modifications in the amount, number of shares or manner of reduction of paid up share capital. 195 Following the receipt of the Government Approval and in pursuance of the powers granted to the Board by the shareholders of our Bank in the extraordinary general meeting dated September 11, 2004, our Board resolved by resolution dated January 13, 2005 that the amount raised through the issue of 30,000,000 Equity Shares out of the total issue of 80,000,000 Equity Shares would be returned to the Government by March 31, 2005, and that the Bank would reduce 30,000,000 Equity Shares held by the President of India in accordance with the provisions of Section 3(2BBA)(a) of the Banking Companies Acquisition Act 1970. Accordingly, we shall transfer the amount raised through the issue of 30,000,000 Equity Shares out of the total issue of 80,000,000 Equity Shares to the Government by March 31, 2005. For further details, see sections titled “Risk Factors” on page x, “General Information - Authority for the Issue” on page 7, “Capital Structure” on page 22 and “Government Approvals” on page 182 and for details on Section 3(2BBA)(a) of the Banking Companies Acquisition Act 1970, see the section titled “Main Provisions of Constitutional Documents” on page 248. Except as disclosed above, in the opinion of the Board of our Bank, there have not arisen, since the date of the last financial statements disclosed in this Red Herring Prospectus, any circumstances that materially or adversely affect or are likely to affect the profitability of our Bank taken as a whole or the value of its consolidated assets or its ability to pay its material liabilities within the next twelve months. IV. Against Our Group Companies A. Litigation against our subsidiaries A.1 PNB Housing Finance Limited 1. Contingent Liabilities not provided for as of September 31, 2004: Rs. 30,396,759. 2. Litigation against PNB Housing Finance Limited as on February 7, 2005: Sl. No. Name and No. Nature of dispute Amount Involved Status of case 1. Interest Tax Income Tax Department has raised demand Appeal pending in ITAT in respect of interest tax on securities for these financial years, which have been duly paid. 93-94 Rs. 494,618 94-95 Rs. 1,420,334 95-96 Rs. 1,336,233 96-97 Rs. 875,911 2. Income Tax Income Tax Department has raised demand Rs. 29,655,591 Appeal pending in ITAT for financial year 95-96, which has been duly paid, on the ground that a write off in GDA account of Rs. 54,235,290 has not been credited in GDA’s account. 3. Income Tax Income Tax Department has raised demand Rs. 13,622,902 Appeal pending in ITAT for financial year 96–97, which has been duly paid, in respect of computation u/s 36(1)(viii) by allowing claim of only Rs. 410,876 against Rs. 23,156,460 claimed by us. 4. Income Tax Income Tax Department has gone into appeal Rs. 1,035,731 Consequential impact in respect of financial year 97-98, which has of appeal pertaining to been duly paid by withdrawing the MAT financial year 96-97 is credited of financial year 96-97 allowed in pending in ITAT. the financial year 97-98. 196 Sl. Name and No. Nature of dispute Amount Involved Status No. of case 5. Income Tax Income Tax Department has gone into appeal Rs. 5,011,687 Appeal pending in ITAT. in respect of financial year 97-98 on the ground that common expenses should be allocated to interest on tax free bonds for computation of deduction under section 36 (1)(viii) of the IT Act. 6. Income Tax Income Tax Department has raised a demand Rs. 5,00,000 PNB Housing Finance in respect of financial year 2001-02 by Limited to file an appeal disallowing deferred revenue expenditure against the said order in and expenditure in respect of tax-free the ITAT. income. A.2 PNB Gilts Limited 1. Contingent Liabilities not provided for as of September 30, 2004: Rs.0.377 million 2. Litigation against as of February 7, 2005 : Nature of Litigation/ Quantum of claims/ Status/outcome of litigation, dispute Dispute/ Default/Non- demands payment Interest Tax 310,434 A sum of Rs. 31,77,908/- had been paid to the income tax authorities as interest tax for the year 1996-97 against which the company sought refund. The appellate authorities have adjudicated in favour of the company and a refund of Rs. 28.67 lacs has been received along-with interest of Rs. 18.86 lacs. The company has preferred appeal for the remaining amount of Rs. 3.10 lacs. The last appeal has not come up for hearing so far. Mafatlal Consultancy 400,000 The company had filed a case against Mafatlal Consultancy Services (MCS) for Services for recovery of dues in the Delhi Court. The case was breach of contract that decreed in favour of PNB Gilts Limited. The decree has since been was executed for transferred to Mumbai District Court for execution through M/s. development of D’Lima and Associates, Advocates, High Court Mumbai on August integrated software 8, 2001. However, it has been informed that even though around package. 3 years have elapsed since the decree was transferred to Mumbai, the advocates have not been able to obtain a clarification of the assets of Mafatlal Consultancy Services Ltd. and whether the same can be attached for effectively satisfying the decree. Efforts are being made for the attachment of the assets through our advocate for realization of our dues. In the past, no negative action/penalties have been imposed by any regulatory authority. In addition, a complaint has been filed under Indian Penal Code against M/s. Delfin Finance Ltd, in which Mr. Mohanjit Singh, one of our directors is director, regarding pre-emptive purchase of property under provisions of the IT Act. 197 A.3 PNB Asset Management Company Limited 1. Contingent Liabilities not provided for as of March 31, 2004: Rs. 299,000 2. Litigation against as of February 7, 2005: In the past, no negative action/penalties have been imposed by any regulatory authority other than mentioned below: (a) SEBI had referred a case of two schemes to an adjudication officer in which the company exceeded the 10% investment limit (in single scrip) of the Net Asset Value. The adjudication officer levied a penalty of Rs. 0.2 million, which was paid by the PNB Asset Management Company Limited on May 9, 2001. (b) The IT Department has raised a demand of Rs. 17.05 lacs for the assessment year 2002-2003 vide letter dated November 22, 2004. PNB Asset Management Company Limited has filed an appeal with the Commissioner of Income Tax (Appeals). B. Litigation against our associates B1. M/s. Assets Care Enterprises Ltd. 2. Contingent Liabilities not provided for as of September 30, 2004: NIL. 3. Litigation and penalties filed against the company as on February 7, 2005: NIL. B 2. UTI Asset Management Company (P) Limited. 1. Contingent Liabilities not provided for as of March 31, 2004: NIL 2. Litigation and penalties filed against the company as on December 31, 2004: Sr. Forum/ Name and No. of the case Nature of dispute Amt. Involved No. 1. CGIT, Mumbai – George & Modern Contract workmen engaged in Nil Maintenance Services vs. UTI (CGIT 76 of cleaning services seeking regulation 2004 arising out of W.P. No. 61 of 1996) in UTI 2. CGIT, Mumbai – Property Guards Vs. UTI Contract workmen engaged in Nil (CGIT 77 of 2004 arising out of W.P. No. security services seeking 3435 of 1990) regularization in UTI 3. CGIT, Mumbai – Property Guards Vs. UTI Contract workmen engaged in Nil (CGIT 78 of 2004 arising out of W.P. No. security services seeking 1165 of 1991) regularization in UTI 4. CGIT, Mumbai, George & Modern Contract workmen engaged in Nil Maintenance Services Vs. UTI (CGIT 79 of cleaning services seeking 2004 arising out of W.P. No. 2129 of 1999) regularization in UTI 5. CGIT, Mumbai – Property Guards Vs. UTI Contract workmen engaged in Nil (CGIT 80 of 2004 arising out of W.P. No. security services seeking 2017 of 1992). regularization in UTI 6. Mumbai II.C., Precipe in W.P. Nos. 2017 of Contract workmen engaged in Nil 1992, 3435 of 1990 & 1165 of 1991 security services seeking extension of interim relief till Judge is appointed at CGIT 198 Sr. Forum/ Name and No. of the case Nature of dispute Amt. Involved No. 7. Mumbai II.C., Shantaram B. Ghadi & Ors. Contract workmen engaged in Nil Vs. UTI W.P. No. (Lodging) 2708 of 2004 cleaning services praying to extend (related to W.P. No. 61 of 1996) the interim relief until such an application in CGIT No. 76 of 2004 is decided. 8. Supreme Court – Shri Digvijay Singh Vs. Intended appeal on the order of Nil UTI Mumbai H.C. refusing to stay the disciplinary enquiry on the ground that criminal proceedings are pending. 9. Delhi H.C. – Shri Atul Kumar Jain Vs. UTI Seeking not to be relieved under Nil (W.P. No. 8778 of 2004) VSS as he has withdrawn his VSS application 10. CGIT Mumbai – Shri S.R. Keer Vs. UTI (CGIT Dismissed employee seeking Rs. 18 lacs No. 1/25 of 2004) reinstatement on the ground that he (Assistant has been acquitted subsequently by dismissed w.e.f. criminal court on same charges 13.02.1986) 11. CGIT Mumbai – BKKM Vs. UTI (CGIT No. 1/ Contract workmen engaged in Nil 48 of 2004) cleaning/security services seeking regularization in UTI 12. CGIT Mumbai – AIUTEA Vs. UTI (CGIT No. ATUTEA seeking restoration of Nil 1/20 of 2004) additional comm. For P.F. etc. 13. CGIT Mumbai – AIUTEA Vs. UTI (CGIT No. ATUTEA seeking for 3rd pension Rs. 17.5 crores (The 1/26 of 2004) option option is effective from 2000. The liability is towards additional funding required in respect of left over Class III and Class IV staff based on the average earnings and average service). 14. Delhi H.C. – Shri A.K. Anand Vs. UTI (W.P. Seeking promotion to Gr. ‘C’ Rs. 50,000/- No. 1067 of 1999) (IR-3-35) retrospectively from 01.01.95. However, he has been subsequently promoted to Gr. ‘C’ on 10.05.1999. 199 Sr. Forum/ Name and No. of the case Nature of dispute Amt. Involved No. 15. Delhi H.C. – Shri A.K. Anand Vs. UTI (WP Seeking stay of his deputation to Nil No. 6567 of 2002) (The earlier W.P. No. 1369 UTISI, Navi Mumbai of 2002 has been disposed off) 16. Guwahati Civil Court – Shri Mukesh Kapoor Seeking to consider his Promotion Nil Vs. UTI (Title Suit No. 31 of 2004) to Gr. ‘B’ 17. Allahabad H.C. – Shri Shyamji Mishra & ors. Ex-temporary employee seeking Nil Vs. UTI (WP No. 32954 of 1990) regularization as permanent employee 18. CGIT, Delhi – Shri J.C. Katyal Vs. UTI (ID Dismissed employee challenging Rs. 25 lacs No. 2 of 1992) his dismissal (Assistant dismissed in 1987) 19. Mumbai R.C. – UTI Vs. S.B. Vichare (W.P. UTI challenging the award of the Rs. 18 lacs No. 2311 of 1999) Tribunal ordering reinstatement (Assistant dismissed in 1983) 20. Kolkata H.C. – Shri Ashok Kumar Das Vs. Wait listed employee who could not Nil UTI (C.O. No. 4546 of 1996) be offered employment due to lack of vacancies. Case filed seeking appointment. 21. Kolkata H.C. – Shri Prasanna (Orang Vs. UTI Wait listed employee who could not Nil (C.O. No. 11219 (W) of 1996) be offered employment due to lack of vacancies. Case filed seeking appointment. 22. Kolkata H.C. – Shri Tannjendra Mukherjee Wait listed employee who could not Nil Vs. UTI (W.P. No. 1005 of 1997) be offered employment due to lack of vacancies. Case filed seeking appointment. 23. Patna H.C. – Shri Ravi S. Prasad Vs. UTI Wait listed employee who could not Nil (MJC 3208 of 1998) (W.P. No. 9269 of 2001) be offered employment due to lack of vacancies. Case filed seeking appointment. 24. Hyderabad H.C. – Shri Yani Prasad Vs. UTI Wait listed employee who could not Nil (W.P. No. 13104 of 2001) be offered employment due to lack of vacancies. Case filed seeking appointment. 25. Ahmedabad Civil Court – Shri D.M. Kalwala Wait listed employee who could not Nil Vs. UTI (C.S. No. 2126 of 1997) be offered employment due to lack of vacancies. Case filed seeking appointment. 200 Sr. Forum/ Name and No. of the case Nature of dispute Amt. Involved No. 26. Mumbai H.C. – BKKM Vs. UTI (W.P. No. 4925 Contract workmen engaged in Nil of 2000) Electrical/AC maintenance seeking abolition of contract labour system 27. Mumbai H.C. – Contract Laghu Udyog Canteen contract workmen seeking Nil Kamgar Union Vs. UTI (W.P. No. 2908 of quashing of the order of Tribunal 2003) holding that they are not entitled for regularization in UTI 28. Mumbai H.C. – UTI Vs. State of Maharashtra Appeal of UTI against order of the Nil (W.P. No. 4078 of 2000) Magistrate to engage security guards through the Board 29. Madras H.C. – UTI Vs. UT SC/ST Obtaining injunction against Nil Association, Chennai (Civil Suit No. 199 demonstration on March 16, 1999. of 1999) 30. Madras H.C. – Shri Selvaraj, UT SC/ST SC/ST Assn. Praying that engaging Nil Assn. Chennai Vs. UTI (W.P. No. 24611 of VSS optees at UTI/SL without 2003) notification to employment exchange etc. as improper. nd 31. 22 Metropolitan Magistrate, Andheri, Reg. Non display of notices under Nil Mumbai LEO Vs. UTI (CC No. 120/SLC/ Contract Labour Act 2003) 32. Magistrate court, Kolkata LEO Vs. UTI (R.K. Non – display of notices under Nil Mangla) No. 3465 of 2003) Contract Labour 33. Magistrate court, Kolkata LEO Vs. UTI (S.K. Regarding not obtaining registration Nil Saha) (No. 3888 of 2003) certificate under Contract Labour Act. 34. Percipies No. _____ of 2004 in W.P. No. 2017 Filed by contract workmen (Security Nil of 1992, W.P. No. 3435 of 1990, W.P. 1165 Guards) to extend interim relief in of 1991. Mumbai H.C. view of non posting of Judge in CGIT. 35. W.P. No. 19854 of 2004. Yogesth Singh Vs. Assistant Manager, Patna Office, Union of India and Ors. Delhi H.C. praying to release salary for his absence periods, declare the enquiry as illegal, considering of promotion etc. 36 Civil Suit No. 48 of 2004, Manmohan VSS employee praying that his Bamania Vs. UTI AMC, District Court, release under VSS is illegal and for Jodhpur reinstatement. 37. CGIT Mumbai ______of 2005, contract The canteen services discontinued labour Udyog Kamgar Union Vs. UTI AMC, from 25.02.2004. The 39 canteen canteen case (Payment of legal dues) workmen praying for final legal dues. 201 B 3. UTI Trustee Company (P) Limited. 1. Contingent Liabilities not provided for as of March 31, 2004: NIL 2. Litigation and penalties filed against the company as on December 31, 2004 are as follows: A. Criminal cases. Sr. No. Nature of Case No. Name of the Complainant Name of the Court Litigation / Dispute / Default / Non Payment 1 Criminal Criminal Revision Naresh Kumar Sethi District and Sessions Judge, Jaipur Petition 66 2 Criminal Criminal Revision Naresh Kumar Sethi District and Sessions Judge, Jaipur Petition 67 3 Criminal Criminal Revision Naresh Kumar Sethi District and Sessions Judge, Jaipur Petition 68 4 Criminal 963/98 D.B. Dutta Judicial Magistrate -1, Patna 5 Criminal 501/97 K.K.P. Verma Judicial Magistrate-1, Muzaffarpur 6 Criminal 41/97 Shyam Sunder Kejriwal CIM, Muzaffarpur 7 Criminal 142/98 Kiran Devi Judicial Magistrate -1, Patna 8 Criminal 421/99 Ranjeet Kumar Judicial Magistrate-1, Patna 9 Criminal 482/97 Krishna Kumar Kejriwal Judicial Magistrate-1, Bhagalpur 10 Criminal 195/2000 Nawal Kishore Prasad CIM, Siwan 11 Criminal 44/99 Suresh Kumar Singh SDIM Hajipur 12 Criminal 407/96 Deobrat Singh CIM Dhanbad 13 Criminal 1316/2000 Bhagwan CIM Dhanbad 14 Criminal CR. Misc 9008/99 Deobrat Singh High Court, Ranchi 15 Criminal 160/98 (03.04.98) Sanjeev Bindal CIM, Kasganj 16 Criminal 1034/98 Vijay Kumar Judicial Magistrate-1, Purnea 17 Criminal CR Case No.954/ Kailash Aggarwal Judicial Magistrate, Jamshedpur 97 18 Criminal Case No. 8670/98 Jitendra Kumar CIM, Muzaffarnagar 202 Sr. No. Nature of Case No. Name of the Complainant Name of the Court Litigation / Dispute / Default / Non Payment 19 Criminal Case no.94/97 Teg Bahadur ACJM-II, Muzaffarnagar 20 Criminal 879/97 Virendra Singh ACJM, Muzaffarnagar 21 Criminal 1954/95 G.J.Suryawala JM (FC), Baroda 22 Criminal 1239/01 R.Singh SDJM, Jamshedpur 23 Criminal 9876/2002 Pratima Sarkar CJM-I B. Consumer Cases Place Number of Cases Western Zone 642 Northern Zone 97 Eastern Zone 110 Southern Zone 28 Total 877 B 4. Principal PNB Asset Management Company Private Limited. 1. Contingent Liabilities not provided for as of September 30, 2004: Rs. 140,215. 2. Litigation and penalties filed against the company as on February 7, 2005: The total liability of the company with regard to its litigation does not exceed Rs 500,000. B 5. Principal Trustee Company Private Limited. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation and penalties filed against the company as on February 7, 2005: NIL. B 6. Everest Bank Limited 1. Contingent Liabilities not provided for as of September 30, 2004: The bank does not have any statutory tax liability except a contingent liability to the tune of Nepali Rupaiya 2,855,953 in relation to the cases where the bank has filed an appeal against the tax assessment orders for 3 years as mentioned below. 2. Litigation against as on February 7, 2005: No civil or criminal cases and no cases against directors and employees. Income tax/Interest tax matters: Everest Bank Limited has filed appeals against the decisions of the Inland Revenue Department in which they opened its income tax assessment files for the three years, i.e. 1997-98, 1999-2000, 200-01. While opening the files they have added back the residual amount after payment of statutory bonus which had been transferred to the bank’s Employees Provident Fund as per law. 3. No penalties have been imposed by regulatory bodies against the bank in the last three years. 203 B7 PNB Principal Financial Planners Limited 1. Contingent Liabilities not provided for as of February 7, 2005: Nil 2. Litigation and penalties filed against the company as on February 7, 2005: Nil B8 PNB Principal Insurance Advisory Services Private Limited 1. Contingent Liabilities not provided for as of February 7, 2005: Nil 2. Litigation and penalties filed against the company as on February 7, 2005: Nil PNB Principal Insurance and Pension Company Limited is yet to be incorporated. C. Litigation against RRBs: C.1 Bhojpur- Rohtas Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Service Matters: There are 38 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal and one case pending before the Supreme Court at various stages of hearing and disposal. B. Criminal cases: There are 7 cases in which the Bhojpur- Rohtas Gramin Bank has been named a respondent in a criminal appeal. These cases relate to cases in which Bhojpur- Rohtas Gramin Bank had filed criminal complaints against certain persons including employees and these persons have filed appeals against the orders passed by lower courts against them and named Bhojpur- Rohtas Gramin Bank as a respondent. C 2. Magadh Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation and penalties filed against the company as on February 5, 2005 are as follows: There are 18 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal. C 3. Nalanda Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: There are 21 cases relating to service and labour matters pending before various forums at various stages of hearing and disposal. C 4. Patliputra Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: Bank guarantee of Rs. 0.676 million. 2. Litigation and penalties filed against the company as on February 5, 2005: NIL. C 5. Haryana Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: There are 34 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 6. Hissar Sirsa Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: 204 A. Service Matters: There are 9 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 7. Ambala Kurukshetra Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Consumer case: S. No. Name & No. of Case Nature of Dispute Amount Involved Status 1. B.O. Lakhnaura – Fraud detected Rs. 1.396 million Pending with Consumer 21 cases filed by in branch in 1999 court. different parties in However, an amount of consumer court Rs. 2.07 million is against the bank. outstanding in books of accounts as provision to meet out this liability as well as others. B. Service Matters: There are 19 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 8. Himachal Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: S.No. Name and No. of case Nature of dispute Amount involved Status (Amount in Rs.) 1. 1 civil case Deposit claim 22,000 Pending in the court 2. 21 employee Seniority/promotion NIL Pending in the court related cases 3. 3 Income tax/interest Tax exemption 22,956,000 Appeal with Income tax tax matters Commissioner & Himachal Pradesh High Court. C 9. Shivalik Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Disputes/Litigations towards Tax liability: Shivalik Kshetriya Gramin Bank, being a RRB, are claiming the exemption under Sec 80 P (2)(a)(i) of IT Act for the profit earned. But income tax officer, Hoshiarpur while assessing the Income Tax Returns, has not allowed the deduction in respect of interest on investments by treating it as the income from other sources. The income tax officer, Hoshiarpur has so far opened the cases for assessment years 1996-97 to 2001-02. The assessment for the assessment year 2001-02 is still in progress whereas the assessment for the financial year 1996-97 to 2000-01 was completed before 31.03.2004. Shivalik Kshetriya Gramin Bank has 205 deposited the demand raised by income tax officer, Hoshiarpur before 31.03.2004. The assessment year wise detail is as under: Assessment Demand Amount Date of Appeal pending with year raised deposited Deposit 1996-1997 5,983,007 5,983,007 11.02.2000 Appellate Tribunal, Amritsar 1997-1998 3,952,128 3,952,128 11.02.2000 Appellate Tribunal, Amritsar 1998-1999 16,819,991 16,819,991 18.03.2002 CIT (Appeal), Jalandhar 1999-2000 23,895,015 23,895,015 19.09.2003 CIT (Appeal), Jalandhar 2000-2001 31,281,093 31,281,093 25.11.2003 CIT (Appeal), Jalandhar Total 81,931,234 81,931,234 The demand raised by assessing officer for the assessment year 2001-2002 is Rs.27,511,398 which was to be deposited before November 24, 2004 and the assessment of the returns for the later years has not been yet reopened by income tax officer, Hoshiarpur. The amount has been deposited without booking the expenditure. The amount is being paid to the debit of income tax paid account and is shown in the balance sheet under Schedule 11 (item III). The amount has been shown as disputed tax liability as the appeals are pending at various levels. B. Cases relating to employees and service matters: There are 5 cases relating to service and labour matters pending before various forums, including the state high court at various stages of hearing and disposal. C 10. Kapurthala –Firozpur Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: 1.54 million. 2. Litigation against as of February 5, 2005 are as follows: There are 11 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 11. Gurdaspur Amritsar Kshetriya Gramin Vikas Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: 1.078 million. 2. Litigation against as of February 5, 2005 are as follows: There are 15 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 12. Shekhawati Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows : There are 38 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 13. Alwar Bharatpur Anchalik Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: 1.110 million. 2. Litigation against as of February 5, 2005 are as follows: There are 31 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. 206 C 14. Kisan Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Service matters: There are 13 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. B. Criminal Cases: There are 2 criminal cases pending before various forums at various stages of hearing and disposal. C 15. Devi Patan Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Service Matters There are 23 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. B. Criminal Cases: There are 2 cases in which the Devi Patan Kshetriya Gramin Bank has been named a respondent in a criminal appeal. These cases relate to cases in which Devi Patan Kshetriya Gramin Bank had filed criminal complaints against certain persons including employees and these persons have filed appeals against the orders passed by lower courts against them and named Devi Patan Kshetriya Gramin Bank as a respondent. C 16. Rani Lakshmibai Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: 0.927 million. 2. Litigation against as of February 5, 2005 are as follows: There are 38 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 17. Vidur Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: There are 41 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 18. Muzaffarnagar Kshetriya Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: There are 6 cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. C 19. Hindon Gramin Bank. 1. Contingent Liabilities not provided for as of September 30, 2004: NIL. 2. Litigation against as of February 5, 2005 are as follows: A. Civil cases There are 3 civil cases relating to service and labour matters pending before various forums, including the state high court, the industrial tribunal at various stages of hearing and disposal. B. Criminal case: There is 1 criminal case against the bank pending before judicial magistrate, Ghaziabad. 207 DIVIDEND POLICY The declaration and payment of dividends will be recommended by our Board of Directors, at their discretion, and will depend on a number of factors, including but not limited to our profits, capital requirements and overall financial condition, and shall be subject to the guidelines issued by RBI. For further details on restrictions on dividend declaration, refer to the section titled “Regulations and Policies – Restrictions on Payment of Dividends” on page 177. The dividends declared by our Bank during the last five fiscal have been presented below. Year ended March 31, Fiscal Fiscal Fiscal Fiscal Fiscal 2004 2003 2002 2001 2000 Face value of Equity Shares (in Rs. per share) 10 10 10 10 10 Dividend (in Rs. Million) 1061.2 928.5 636.7 530.7 500.0 Dividend Tax (%) 13.6 11.9 Nil 5.41 6.76 Dividend per Equity Share (Rs) 4.00 3.50 3.00 2.50 2.36 Dividend Rate Approx. (%) 40.00 35.00 30.00 25.00 23.56 In addition our Board has declared interim dividend at the rate of 30%, i.e. Rs. 3 per Equity Share of face value Rs. 10, by its resolution dated January 28, 2005. The record date for the same is February 28, 2005. The amounts paid as dividends in the past are not necessarily indicative of our dividend policy or dividend amounts, if any, in the future. Future dividends will depend upon our revenues, profits, cash flow, financial condition, capital requirements and other factors. 208 OTHER REGULATORY DISCLOSURES Stock Market Data For Our Equity Shares See the section titled “History and Certain Corporate Matters - Stock Market Data” on page 65. Particulars Regarding Public Issues During The Last Five Years Details of the public issues made by our Bank in the previous five years are as follows: Public Issue of Equity Shares (April 2002) Public Issue in which 53,061,200 Equity Shares of Rs. 10 each for cash at a premium of Rs. 21 per Equity Share aggregating Rs. 1,644.9 million were allotted to public. Opening Date March 21, 2002 Closing Date March 28, 2002 Date of Allotment April 20, 2002 Date of Refunds April 20, 2002 Date of Listing on Stock Exchanges BSE on April 25, 2002 NSE on April 24, 2002 DSE on April 24, 2002 We had applied for voluntary delisting of our Equity Shares from the DSE vide letter dated October 5, 2004 pursuant to a shareholder resolution dated July 3, 2004. We have received a letter dated October 13, 2004 bearing reference number DSE/ LIST/R/96 from the DSE informing the Bank that the securities of the Bank are delisted from the DSE with immediate effect. Promise vs. Performance We have not made any projections in the offer document of our previous capital issues during the last five years. The funds raised from these capital issues have been utilised for our business as mentioned in the offer document for the same. Servicing Behaviour There has been no default in payment of statutory dues or of interest or principal in respect of our borrowings or deposits. Companies Under The Same Management There are no companies under the same management. Mechanism For Redressal Of Investor Grievances The agreement between the Registrar to the Issue and us, will provide for retention of records with the Registrar to the Issue for a period of at least one year from the last date of dispatch of letters of Allotment, demat credit, refund orders to enable the investors to approach the Registrar to the Issue for redressal of their grievances. All grievances relating to the Issue may be addressed to the Registrar to the Issue, giving full details such as name, address of the applicant, application number, number of shares applied for, amount paid on application, Depository Participant, and the respective Syndicate Member or collection center where the application was submitted. Disposal Of Investor Grievances We estimate that the average time required by us or the Registrar to the Issue for the redressal of routine investor grievances shall be 15 days from the date of receipt of the complaint. In case of non-routine complaints and complaints where external agencies are involved, we will seek to redress these complaints as expeditiously as possible. 209 The details of the investor grievances are as follows: Category Pending on Received during Processed during Pending on December 1, 2004 December 2004 December 2004 January 1, 2005 Equity Shares NIL 138 138 NIL We have appointed Ms. Malathi Mohan, Company Secretary as the Compliance Officer and she may be contacted in case of any pre-Issue or post-Issue-related problems. She can be contacted at the following address: Ms. Malathi Mohan, Punjab National Bank, Share Department, 2nd Floor, 5, Sansad Marg, New Delhi 110 001. Tel: (91 11) 2332 3657 Fax: (91 11) 2371 1663 E-mail: hosd@pnb.co.in 210 STATEMENT OF TAX BENEFITS The following tax benefits shall be available to the Bank and the prospective shareholders under the current Direct Tax laws: A. INCOME TAX TO THE BANK 1. Subject to the provisions of Section 10(34) read with Section 115-O of the Income Tax Act, 1961, income earned by way of dividends from a domestic company and also under Section 10(35), income received in respect of units of Unit Trust of India and units of a mutual fund specified under Section 10(23)(D) are exempt from tax in the hands of the Bank. 2. Under Section 36(1)(viia) of the Income Tax Act, in respect of any provision made for bad and doubtful debts, the Bank is entitled to a deduction not exceeding: (i) 7.5% of the total income (computed before making any deductions under the said clause and Chapter VIA) and (ii) 10% of the aggregate average advances made by the rural branches of the bank computed in the prescribed manner. 3. Apart from the deduction available under Section 36(1)(viia) of the Income Tax Act, the Bank is entitled to claim a deduction under Section 36(1)(vii) of the Income Tax Act for the amount of bad debts written off as irrecoverable in its books of account. The deduction is limited to the amount of such debt or part thereof, which exceeds the credit balance in the provision for bad and doubtful debts account made under Section 36(1)(viia) and further provisions of Section 36(2)(v) have to be complied with. 4. As per Section 43(D) of the Income Tax Act, 1961, interest income on certain categories of bad or doubtful debts as specified in Rule 6EA of the Income Tax Rules, 1962 having regard to the guidelines issued by Reserve Bank of India in relation to such debts shall be chargeable to tax, only in the year in which it is actually received or the year in which it is credited to the Profit & Loss Account by the Bank, whichever is earlier. 5. Under the provisions of Section 112 of the Income Tax Act, 1961, taxable long term capital gains (not covered under Section 10(38) of the Act), arising on transfer of listed securities or units, shall be taxed at a rate of 20% (plus applicable surcharge and education cess) after indexation; or at 10% (plus applicable surcharge and education cess), without indexation, in accordance with and subject to provisions of Section 48 of the Income Tax Act. 6. In accordance with and subject to the provisions of Section 10-23(G) of the Income Tax Act, any income by way of dividends (other than dividend referred to in Section 115-O), interest or long term capital gains of the Bank arising from investments made on or after the first day of June 1998, by way of shares or long terms finance in any specified enterprise wholly engaged in the business of (i) developing (ii) maintaining and operating or (iii) developing, maintaining operating, any specified infrastructure facility and which has been approved by the Central Government and which satisfies the prescribed conditions as per Rule 2E of the Income Tax Rules, 1962, is exempted from tax. Provided that income by way of dividends, other than dividend referred to in Section 115-O, interest or long-term capital gain of an infrastructure company, shall be taken into account in computing the book profit and income tax payable under Section 115 JB. 7. As per the provisions of Section 80-LA of the Income Tax Act where gross total income, in any previous year, includes any income from an offshore banking unit in a special economic zone and from a business with an undertaking located in a special economic zone or any other undertaking which develops, or develops and operates, or operates and maintains a special economic zone, shall subject to the fulfillment of the conditions specified in the said Section 80 LA, be entitled to 100% deduction of such income for three consecutive assessment years, beginning with the assessment year relevant to the previous year in which the Reserve Bank of India’s permission to open the offshore unit shall have been obtained, and after those three years, 50% deduction of such income for the next two consecutive assessment years. 8. As per Sections 54EC and 54ED of the Income Tax Act, subject to the conditions specified therein, tax on capital gains (other than under Section 10 (38)) arising from the transfer of a long-term capital asset in respect of specified securities is exempt from tax, provided that assessee at any time within a period of six months after the date of the transfer, invested the whole of the capital gains in any specified long-term asset for the purposes of Section 54EC or in acquiring shares of a public company offered for subscription by way of a public issue for the purposes of Section 54ED of the Income Tax Act. If only a portion of capital gains is so invested, then the exemption is available proportionately. Further, this is subject to 211 withdrawal of exemption under the circumstances set out in Sections 54EC(2) & 54ED(2). 9. By virtue of Section 10(38) inserted by Finance (No. 2) Act, 2004, income arising form transfer of long-term capital asset, being an equity share in a company or a unit of an equity oriented fund is exempt form tax, if the transaction of such sale has been entered into on or after 1.10.2004 and such transaction is chargeable to securities transaction tax under the Chapter VII of the Finance (No, 2) Act, 2004. 10. By the virtue of Section 111 A inserted by Finance (No. 2) Act 2004, short term capital gain on transfer of equity share in a Company or a unit of an equity oriented fund shall be chargeable to tax @ 10%, if the transaction of such sale has been entered into on or after 1.10.2004 and such transaction is chargeable to securities transaction tax under the Chapter VII of the Finance (No, 2) Act, 2004. B. To the members of the Bank B1. Under the Income Tax Act, 1961 i. All Members 1 By virtue of Section 10(38) inserted by Finance (No. 2) Act, 2004, income arising from transfer of long-term capital asset, being an equity share in the company is exempt from tax, if the transaction of such sale has been entered into on or after 1.10.2004 and such transaction is chargeable to the securities transaction tax under that chapter. 2 By virtue of Section 111 A inserted by Finance (No. 2) Act 2004, short term capital gain on transfer of equity share in the Company shall be chargeable to tax @ 10%, if the transaction of such sale has been entered into on or after 1.10.2004 and such transaction is chargeable to securities transaction tax under that chapter. However, where the income includes any such short-term capital gain, it shall not be considered for deduction under chapter VIA and rebate under Section 88 of Income tax Act, 1961. 3 By virtue of Section 10(34) of the Income Tax Act, income earned by way of dividend income from a domestic company referred to in Section 115O of the Income Tax Act, are exempt from tax in the hands of the shareholders. Further, Section 94(7) of the Income Tax Act provides that loss arising from the purchase and sale of shares purchased within a period of three months prior to the record date and sold within a period of three months after such date, will be disallowed to the extent of dividends on such shares are claimed as tax exempt by the shareholder. ii. To Our Resident Shareholders 1. Under Section 54EC of the Income Tax Act, 1961 and subject to the conditions and to the extent specified therein, long term capital gains (not covered under Section 10 (38) of the Act) arising on the transfer of shares of the Bank will be exempt from capital gains tax if the capital gain are invested within a period of 6 months from the date of transfer in the bonds issued by - National Bank for Agriculture and Rural Development established under Section 3 of the National Bank for Agriculture and Rural Development Act, 1981. - National Highway Authority of India constituted under Section 3 of National Highway Authority of India Act, 1988. - Rural Electrification Corporation Limited, a Company formed and registered under the Companies Act, 1956. - National Housing Bank established under Section 3(1) of the National Housing Bank Act, 1987. - Small Industries Development Bank of India established under Section 3(1) of The Small Industries Development Bank of India Act, 1989. If only part of the capital gain is so reinvested, the exemption shall be proportionately reduced. The amount so exempted shall be chargeable to tax subsequently, if the specified assets are transferred or converted within three years from the date of their acquisition. 2. Under Section 54ED of the Income Tax Act, 1961 and subject to the conditions and to the extent specified therein, long term capital gains (not covered under Section 10 (38) of the Act) arising on the transfer of shares of the Bank, will be exempt from capital gains tax if the capital gain is invested in equity shares of an Indian public company forming part of an eligible public issue, within a period of 6 months after the date of such transfer. If only a part of the capital gain is so reinvested, the exemption shall be proportionately reduced. The amount so exempted shall be chargeable to tax 212 subsequently, if the specified assets are transferred or converted within one year from the date of their acquisition. Where the cost of specified equity shares has been taken into account for the above purpose, a deduction from the amount of income tax with reference to such cost shall not be allowed under Section 88. 3. As per the provisions of Section 54F of the Income Tax Act, 1961, long term capital gains arising (not covered under Section 10 (38) of the Act) in the hand of an individual or a HUF on transfer of shares of the Bank shall be exempt if the net consideration is invested in purchase of a residential house within a period of one year before or two years after the date of transfer or construction of a residential house within a period of three years after the date of transfer. The exemption is available proportionately if only a portion of the net consideration is invested as above. The exemption is subject to other conditions specified in the section. 4. Long-term capital gains would accrue to resident shareholders where the equity shares are held for a period of more than 12 months prior to the date of transfer of the shares. In accordance with and subject to the provisions of Section 48 of the Income Tax Act, in order to arrive at the quantum of capital gains, the following amounts would be deductible from the full value of consideration: (i) Cost of acquisition/improvement of the shares as adjusted by the cost inflation index notified by the Central Government and (ii) Expenditure incurred wholly and exclusively in connection with the transfer of the shares. 5. Under Section 112 of the Income Tax Act, 1961 and other relevant provisions of the Act, long term capital gains (not covered under Section 10 (38) of the Act) arising on transfer of shares in the Company, if shares are held for a period exceeding 12 months shall be taxed at a rate of 20% (plus applicable surcharge and education cess) after indexation as provided in the second proviso of Section 48; or at 10% (plus applicable surcharge and education cess) (without indexation), at the option of the shareholders. iii. To non-resident shareholders including NRIs, OCBs and Flls 1. Under Section 54EC of the Income Tax Act, 1961 and subject to the conditions and to the extent specified therein, long term capital gains (not covered under Section 10 (38) of the Act) arising on the transfer of shares of the Bank will be exempt from capital gains tax if the capital gain are invested within a period of 6 months from the date of transfer in the bonds issued by i. National Bank for Agriculture and Rural Development established under Section 3 of the National Bank for Agriculture and Rural Development Act, 1981. ii. National Highway Authority of India constituted under Section 3 of National Highway Authority of India Act, 1988. iii. Rural Electrification Corporation Limited, a Company formed and registered under the Companies Act, 1956. iv. National Housing Bank established under Section 3(1) of the National Housing Bank Act, 1987. v. Small Industries Development Bank of India, established under Section 3(1) of The Small Industries Development Bank of India Act, 1989. If only part of the capital gain is so reinvested, the exemption shall be proportionately reduced. The amount so exempted shall be chargeable to tax subsequently, if the specified assets are transferred or converted within three years from the date of their acquisition. 2. Under Section 54ED of the Income Tax Act, 1961 and subject to the conditions and to the extent specified therein, long term capital gains (not covered under Section 10 (38) of the Act) arising on the transfer of shares of the Bank, will be exempt from capital gains tax if the capital gain is invested in equity shares of an Indian Public Company forming part of an eligible public issue, within a period of 6 months after the date of such transfer. If only part of the capital gains is so reinvested, the exemption shall be proportionately reduced. The amount so exempted shall be chargeable to tax subsequently, if the specified assets are transferred or converted within one year from the date of their acquisition. Where the cost of specified equity shares has been taken into account for the above purpose, a deduction from the amount of income tax with reference to such cost shall not be allowed under Section 88. 3. As per the provisions of Section 54F of the Income Tax Act, 1961, long term capital gains (not covered under Section 10 (38) of the Act), arising in the hand of an individual on transfer of shares of the Bank shall be exempt if the net 213 consideration is invested in purchase of residential house within a period of one year before or two years after the date of transfer or construction of a residential house within a period of three years after the date of transfer. The exemption is available proportionately if only a portion of the net consideration is invested as above. The exemption is subject to other conditions specified in the Section. 4. As per the provision of the first proviso to Section 48 of the Income Tax Act, capital gains arising from transfer of equity shares acquired by non-resident in foreign currency are to be computed by converting the cost of acquisition / improvement, expenditure incurred wholly and exclusively in connection with such transfer and full value of consideration received or accruing into the same foreign currency as was initially utilized in the purchase of equity shares and the capital gain so computed in such foreign currency shall then be reconverted into Indian currency. Cost indexation benefits will not be available in such cases. 5. Under Section 112 of the Income Tax Act, 1961 and other relevant provisions of the act, long term capital gains (not covered under Section 10 (38) of the Act) arising on transfer of shares in the Company, if shares are held for a period exceeding 12 months shall be taxed at a rate of 20% (plus applicable surcharge and education cess) after indexation as provided in the second proviso of Section 48 or at 10% (plus applicable surcharge and education cess) (without indexation), at the option of the shareholders. (Indexation will not be available if investments are made in foreign currency in accordance with the first proviso to Section 48 of the Income Tax Act, 1961 as stated above) 6. A non resident Indian (i.e. an individual being a citizen of India or person of Indian Origin) has an option to be governed by the provisions of Chapter XIIA of the Income Tax Act, 1961 viz. “Special Provisions Relating to certain Incomes of Non-Residents.” (i) Under Section 115E of the Income Tax Act, 1961, where shares in the Company are subscribed to convertible Foreign Exchange by a Non-Resident Indian, long term capital gains arising to the non-resident on transfer of shares shall (in case not covered under Section 10 (38) of the Act) be concessionally taxed at the flat rate of 10% (plus applicable surcharge and education cess) without indexation benefit (ii) Under provisions of Section 115F of the Income Tax Act, 1961, long term capital gains (not covered under Section 10 (38) of the Act) arising to a non resident Indian from the transfer of shares of the Bank subscribed to in convertible Foreign Exchange shall be exempt from Income Tax if the net consideration is reinvested in specified assets within six months of the date of transfer. If only a part of the net consideration is so reinvested the exemption shall be proportionately reduced. The amount so exempted shall be chargeable to tax subsequently, if the specified assets are transferred or converted within three years from the date of their acquisition. (iii) Under provisions of Section 115G of the Income Tax Act, 1961, it shall not be necessary for a Non Resident Indian to furnish his return of income if his only source of income is investment income or long term capital gains or both arising out of assets acquired, purchased or subscribed in convertible foreign exchange and tax deductible at source has been deducted there from. (iv) Under Section 115H of Income Tax Act, where a NRI becomes assessable as a resident in India, along with his return of Income for that year, he may furnish a declaration in writing to the Assessing Officer under Section 139 of the Income Tax Act to the effect that the provisions of the Chapter-XII-A shall continue to apply to him in relation to income derived from equity shares of the company for that year and subsequent years until such assets are converted into money. (v) Under Section 115-I of the Income Tax Act, 1961, a Non Resident Indian may elect not to be governed by the provisions of chapter XIIA for any assessment year by furnishing the return of income under Section 139 of the Income Tax Act declaring therein that the provisions of this chapter shall not apply to him for that assessment year and if he does so the provisions of this chapter shall not apply to him instead the other provisions of the Act shall apply. 7. As per Section 115AD of the Income Tax Act, 1961 long-term capital gains (other than under section 10(38)) arising on transfer of shares purchased by FIIs, in convertible foreign exchange, are taxable at the rate of 10 % (plus applicable surcharge and education cess). Cost indexation benefits will not be available. 214 8. In accordance with and subject to the provisions of Section 196D(2) of the Income Tax Act, no deduction of Tax at Source will be made in respect of Capital Gains arising from the transfer of the Equity Shares referred to in Section 115 AD from sales proceeds payable to the FIIs. To Mutual Funds As per the provisions of Section 10(23D) of the Income Tax Act, tax exemption is available on income of a mutual fund registered under the Securities and Exchange Board of India Act, 1992 and Regulations made there under, or, mutual funds set up by the public sector banks or public financial institutions/authorized by the RBI and subject to the conditions as the Central Government may specify by notification in the Official Gazette. B2. Under The Wealth Tax Act, 1957 Shares are not treated as assets within the meaning of Section 2(ea) of the Wealth Tax Act, 1957. Accordingly, shares purchased in the issue are not liable to wealth-tax in the hands of the shareholders. B3. Under The Gift Tax Act, 1958 Gifts of the shares of the company made on or after October 1, 1998 are not liable to Gift Tax. Notes: i. All the above benefits are as per the current tax law as amended by the Finance Act, 2004 and will be available only to the sole/first name holder in case joint holders hold the shares. ii. In respect of non-residence taxability of capital gains mentioned above shall be further subject to under the Double Taxation Avoidance Agreements, if any, between India and the country in which the non-resident has fiscal domicile. iii. In view of the individual nature of tax consequences, each investor is advised to consult his/her/their own tax advisor with respect to specific tax consequences of his/her/ their participation in the Scheme. 215 FINANCIAL INDEBTEDNESS Set forth below is a brief summary of our aggregate unsecured borrowings of approximately Rs. 23,428.6 million as on September 30, 2004: Nature of Borrowing/ Amount Outstanding Date of Repayment Interest Security Date (in Rs. million) Rate Series III 3978.6 April 9, 2005 13.95 Unsecured Series IV 600 April 30, 2007 12.00 Unsecured NBL 2000 76.5 June 15, 2005 12.25 Unsecured NBL 2000 23.5 September 1, 2005 12.25 Unsecured Series V 2400 April 12, 2006 11.95 Unsecured Series VI 3000 April 1, 2008 10.00 Unsecured Series VII 1800 April 12, 2007 9.40 Unsecured Series VIII 8.3% 950 April 18, 2009 8.30 Unsecured Series VIII 8.8% 2950 April 18, 2012 8.80 Unsecured Series IX 2650 May 4, 2013 5.80 Unsecured Series X 5000 June 8, 2013 5.90 Unsecured Other Borrowings amounting to Rs. 19,520.2 million as on September 30, 2004: (In Rs. million) Category of Borrowings Amount of Borrowing Borrowings in India Overdrafts 5,942.1 SIDBI Refinance 86.7 NABARD Refinance 376.3 Borrowings Outside India 13,115.1 Total 19,520.2 External Commercial Borrowing We have made an external commercial borrowing of USD 100,000,000 from the Bank of America , N.A., Cre¢dit Agricole Indosuz, Hong Kong Branch, Natexis Banques Populares, Singapore Branch, Raiffeisen Zentralbank Osterrich AG, Singapore Branch with BA Asia Limited as Facility Agent. We have entered into a Credit Agreement dated April 30, 2004 for this purpose. The interest rate on the amounts for each term of the loan is the percentage rate per annum equal to the aggregate of the Margin (which is 0.87% p.a. but could change to 0.85% p.a. if a ratings upgrade occurs) and LIBOR (which is either the British Bankers Association Interest Settlement Rate displayed on the Dow Jones Telerate Screen or if no such rate is available, then the rates as quoted by defined reference banks to leading banks in the London interbank market. 216 TERMS OF THE ISSUE The Equity Shares being offered are subject to the provisions of the Banking Regulation Act, Constitutional Documents, the terms of this Red Herring Prospectus, Prospectus, Bid cum Application Form, the Revision Form, the CAN and other terms and conditions as may be incorporated in the allotment advice and other documents/certificates that may be executed in respect of the Issue. The Equity Shares shall also be subject to laws as applicable, guidelines, notifications and regulations relating to the issue of capital and listing and trading of securities issued from time to time by SEBI, the Government, Stock Exchanges, RBI, and/or other authorities, as in force on the date of the Issue and to the extent applicable. Authority for the Issue Our Board of Directors had authorised a fresh issue of up to 50,000,000 Equity Shares pursuant to a resolution passed at its meeting held on September 11, 2004. Our shareholders subsequently authorised the fresh issue of up to 50,000,000 Equity Shares, by a resolution passed unanimously at the general meeting of our Bank held on October 11, 2004 at New Delhi, subject to the approval of the Government, the RBI, SEBI and other applicable authorities. The shareholder resolution gave the Board the authority and power to accept any modification in the proposal as may be required or imposed by such authorities and as agreed to by the Board. Our Bank applied to the Government on October 14, 2004 for its sanction to a fresh issue of up to 50,000,000 Equity Shares. By its letter bearing number F.No.11/29/2004-BOA dated January 7, 2005, pursuant to Section 3 (2B) (c) of the Bank Acquisition Act, the Department of Economic Affairs, MoF, GoI, conveyed its approval for the Issue on inter alia the terms that the Issue size be increased to 80,000,000 Equity Shares and as a condition to the approval, that the amount raised for 30,000,000 Equity Shares through the public offer should be returned to the Government at the Issue Price by March 31, 2005. Further, in terms of the Government Approval, the proportionate cost of public issue to the extent of 30,000,000 Equity Shares is to be borne by the Government. Following the receipt of the Government Approval and in pursuance of the powers granted to the Board by the shareholders of our Bank in the extraordinary general meeting dated October 11, 2004, our Board, through a resolution dated January 13, 2005, authorized the Issue, subject to the conditions specified in the Government Approval. For further details see sections titled “Risk Factors” on page x, “Capital Structure” on page 22 and “Government Approvals” on page 182. We have received permission from the Foreign Exchange Department, RBI vide their letter no.FE.CO.FID/4998/10.02.40(8435)/ 2004-05 dated February 2, 2005 permitting us to issue shares to NRIs / FIIs with repatriation benefits out of our public Issue of 80,000,000 Equity Shares. The RBI has imposed the condition that post Issue the non resident equity holding in our Bank should not exceed 20% of the paid up capital. The permission is further subject to the conditions laid down by the Government of India in their approval letter no. F.No.11/29/2004-BOA dated January 7, 2005, condition prescribed/stipulated by SEBI in this connection and the terms and conditions for issue of shares as stipulated in the Schedule 1 and 2 to RBI Notification No. FEMA.20/2000-RB dated May 03, 2000. Ranking of Equity Shares The Equity Shares being offered shall be subject to the provisions of our Constitutional Documents and shall rank pari passu in all respects with the existing Equity Shares of the Bank including rights in respect of dividend. The Persons in receipt of Allotment will be entitled to dividend or other benefits, if any, declared by our Bank after the date of Allotment. Face Value and Issue Price The Equity Shares with a face value of Rs. 10 each are being offered in terms of this Red Herring Prospectus at a total price of Rs. [G] per Equity Share. At any given point of time there shall be only one denomination for the Equity Shares. Rights of the Equity Shareholder Subject to applicable laws including, among others, the Bank Acquisition Act, the Bank Regulations, the Bank Scheme and the Banking Regulation Act, the equity shareholders of the Bank shall have the following rights: ● Right to receive dividend, if declared. However the declaration of dividend of the Bank is subject to certain restrictions. Please see the restrictions on the payment of dividend in the section titled “Regulation and Policies - Restrictions on Payment of Dividends” on page 177; 217 ● Right to attend general meetings and exercise voting powers, unless prohibited by law. The Bank Acquisition Act states that no shareholder of the Bank, other than the Central Government shall be entitled to exercise voting rights in respect of the shares held by him in excess of one percent of the total voting rights of all the shareholders of the Bank. However the power of shareholders to exercise voting rights is subject to certain restrictions. For information on restrictions on the power of shareholders to exercise voting rights please see the section titled “Regulation and Policies - Restriction on Share Capital and Voting Rights” on page 177; ● Right to vote on a poll either in person or by proxy; ● Subject to the provisions of Clause 3(2D) of the Bank Acquisition Act and Regulations 17 and 19 of the Bank Regulations, the right of free transferability. However the right of free transferability is subject to certain restrictions. For information on these restrictions please see section titled “Main Provisions of Constitutional Documents” on page 248; and ● Such other rights, as may be available to a shareholder of a listed corresponding new bank under the Banking Regulation Act and our Constitutional Documents and under the listing agreements with the Stock Exchange. However, please note that all rights available to shareholders of a company are not available to shareholders of a new corresponding bank. For information on these restrictions please see section titled “Regulation and Policies - Comparative Table of Rights of Shareholders of Companies Act, 1956 and under regulations applicable to corresponding new banks” on page 151. For a detailed description of the main provisions of our Constitutional Documents dealing, among other things, with voting rights, dividend, forfeiture and lien, transfer and transmission see the section titled “Main Provisions of Constitutional Documents” on page 248. Market Lot and Trading Lot In terms of existing SEBI Guidelines, the trading in the Equity Shares shall only be in dematerialised form for all investors. Since trading of our Equity Shares is in dematerialised form, the tradable lot is one Equity Share. The Equity Shares of the Bank shall be allotted only in dematerialised form. Allotment through this Issue will be done only in electronic form in multiples of one Equity Shares subject to a minimum Allotment of [●] Equity Shares. Jurisdiction Exclusive jurisdiction for the purpose of this Issue is with competent courts/authorities in New Delhi, India. Nomination Facility to the Investor In the nature of the rights specified in Section 109A of the Companies Act, the sole or first Bidder, along with other joint Bidder, may nominate any one Person in whom, in the event of the death of sole Bidder or in case of joint Bidders, death of all the Bidders, as the case may be, the Equity Shares allotted, if any, shall vest. A Person, being a nominee, entitled to the Equity Shares by reason of the death of the original holder(s), shall be entitled to the same advantages to which he or she would be entitled if he or she were the registered holder of the Equity Share(s). Where the nominee is a minor, the holder(s) may make a nomination to appoint, in the prescribed manner, any Person to become entitled to Equity Share(s) in the event of his or her death during the minority. A nomination shall stand rescinded upon a sale/ transfer/ alienation of Equity Share(s) by the Person nominating. A buyer will be entitled to make a fresh nomination in the manner prescribed. Fresh nomination can be made only on the prescribed form available on request at the Head Office or at the Registrar and Transfer Agents of our Bank. In the nature of the rights stated in Section 109B of the Companies Act, any Person who becomes a nominee in the manner stated above, shall upon the production of such evidence as may be required by the Board of Directors, elect either: a. to register himself or herself as the holder of the Equity Shares; or b. to make such transfer of the Equity Shares, as the deceased holder could have made. Further, the Board of Directors may at any time give notice requiring any nominee to choose either to be registered himself or herself or to transfer the Equity Shares, and if the notice is not complied with, within a period of 90 days, the Board of Directors may thereafter withhold payment of all dividends, bonuses or other monies payable in respect of the Equity Shares, until the requirements of the notice have been complied with. 218 Notwithstanding anything stated above, since the Allotment in the Issue will be made only in dematerialised mode, there is no need to make a separate nomination with us. Nominations registered with the respective depository participant of the applicant would prevail. If the investors require to change the nomination, they are requested to inform their respective depository participant. Subscription by Non Residents, Eligible NRI, FIIs It is to be distinctly understood that there is no reservation for any Non-Residents, Eligible NRIs, FIIs and such applicants will be treated on the same basis with other categories for the purpose of allocation. As per the RBI regulations, OCBs cannot participate in the Issue. Further, NRIs who are not Eligible NRIs, foreign venture capital funds are not permitted to participate in this Issue. The Equity Shares have not been and will not be registered under the US Securities Act, 1933 (“the Securities Act”) or any state securities laws in the United States and may not be offered or sold within the United States or to, or for the account or benefit of, “U.S. persons” (as defined in Regulation S under the Securities Act), except pursuant to an exemption from, or in a transaction not subject to, the registration requirements of the Securities Act. Accordingly, the Equity Shares are only being offered and sold (i) in the United States to “qualified institutional buyers”, as defined in Rule 144A of the Securities Act, and (ii) outside the United States to certain Persons in offshore transactions in compliance with Regulation S under the Securities Act and the applicable laws of the jurisdiction where those offers and sales occur. 219 ISSUE STRUCTURE The present Issue of 80,000,000 Equity Shares comprising of Net Issue to the public of at least 64,000,000 Equity Shares, a reservation for Employees of up to 8,000,000 Equity Shares and a reservation for Existing Shareholders of up to 8,000,000 Equity Shares, at a price of Rs. [ ] for cash aggregating Rs. [ ] million is being made through the Book Building Process. Employees Existing Shareholders QIBs Non-Institutional Retail Individual Bidders Bidders Number of Up to Up to 8,000,000 Up to 32,000,000 Minimum of 9,600,000 Minimum of Equity 8,000,000 Equity Shares. Equity Shares or Net Equity Shares or Net 22,400,000 Equity Shares Equity Issue less allocation to Issue less allocation to Shares or Net Issue Shares. Non-Institutional QIB Bidders and Retail less allocation to QIB Bidders and Retail Individual Bidders. Bidders and Non- Individual Bidders. Institutional Bidders. Percentage of Up to 10% of Up to 10% of size of Up to 50% of Net Issue Minimum 15% of Net Minimum 35% of Net Issue size size of the the Issue . or Net Issue less Issue or Net Issue less Issue or Net Issue less available for Issue . allocation to Non- allocation to QIB allocation to QIB Bidders allocation Institutional Bidders Bidders and Retail and Non Institutional and Retail Individual Individual Bidders. Bidders. Bidders. Basis of Proportionate. Proportionate. Discretionary. Proportionate. Proportionate. Allocation if respective category is oversubscribed Minimum [ ] Equity [ ] Equity Shares Such number of Such number of [ ] Equity Shares and in Bid# Shares and and in multiples of [ ] Equity Shares that the Equity Shares that multiples of [ ] Equity in multiples Equity Shares Bid Amount exceeds the Bid Amount Share thereafter . of [ ] Equity thereafter. Rs 100,000 and in exceeds Rs 100,000 Shares multiples of [ ] Equity and in multiples of [ ] thereafter. Shares thereafter. Equity Shares thereafter. Maximum Bid Such number Such number of Such number of Such number of Such number of of Equity Equity Shares not Equity Shares not Equity Shares not Equity Shares Shares such exceeding 8,000,000 exceeding the Net exceeding the Net whereby the Bid that the total number of Equity Shares, when Issue, subject to Issue subject to Amount does not Equity applying in the applicable limits. applicable limits. exceed Rs. 100,000. Shares Bid Existing for does not Shareholders exceed 500 Reservation Portion. Equity Shares. Mode of Compulsorily Compulsorily in Compulsorily in Compulsorily in Compulsorily in Allotment in dematerialised mode. dematerialised form. dematerialised form. dematerialised form. dematerialized mode. Trading Lot One Equity One Equity Share. One Equity Share. One Equity Share. One Equity Share. Share . 220 Employees Existing Shareholders QIBs Non-Institutional Retail Individual Bidders Bidders Who can Employees Existing Public financial Eligible NRIs, Resident Individuals (including Apply *** as on Shareholders as on institutions, as Indian individuals, HUFs, Eligible NRIs) January 1, February 11, 2005 specified in Section HUF (in the name of applying for Equity 2005 and who hold Equity 4A of the Companies Karta), companies, Shares such that the Shares of upto Rs. Act, scheduled corporate bodies, Bid Amount does not 100,000 determined commercial banks, scientific institutions exceed Rs. 100,000 in on the basis of the mutual funds, foreign societies and trusts. value. closing price of institutional investors Equity Shares in the registered with SEBI, NSE on February 10, multilateral and 2005. bilateral development financial institutions, and State Industrial Development Corporations, permitted insurance companies registered with the Insurance Regulatory and Development Authority, provident funds with minimum corpus of Rs. 250 million (as permitted by applicable law) and pension funds with minimum corpus of Rs. 250 million in accordance with applicable law. Terms of Margin Margin Amount Margin Amount Margin Amount Margin Amount Payment Amount applicable to Existing applicable to QIB applicable to Non applicable to Retail applicable to Shareholders at the Bidders at the time of Institutional Bidders Individual Bidders at Employees time of submission of submission of Bid at the time of the time of submission at the time of Bid cum Application cum Application Form submission of Bid of Bid cum Application submission Form to the to the Syndicate cum Application Form to the Syndicate of Bid cum Syndicate Members. Members. Form to the Members. Application Syndicate Members. Form to the Syndicate Members. Margin Full Bid Full Bid Amount on Nil. Full Bid Amount on Full Bid Amount on Amount Amount on Bidding. Bidding. Bidding. Bidding. * Subject to valid Bids being received at or above the Issue Price. Under-subscription, if any, in any portion, would be allowed to be met with spillover from any other portions at the discretion of the Bank, in consultation with the BRLMs. 221 # The minimum number of Equity Shares for which Bids can be made by Bidders and the multiples of Equity Shares in which the Bids can be made, shall be advertised at least one day prior to the Bid Opening Date/Issue Opening Date, in Business Standard, an English language newspaper with wide circulation and Hindustan, a Hindi language newspaper with wide circulation. ** An undersubscription for Equity Shares, if any, reserved for Employees and Existing Shareholders would be included in the Net Issue to the public and first be distributed equally between the Retail Portion and the Non-Institutional Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. In the event that the demand in either of the Retail Portion or the Non-Institutional Portion has been met, the Equity Shares shall be allocated to a category in which the demand has not been met. The remaining undersubscribed Equity Shares, if any, after allocation to the Retail Portion and the Non-Institutional Portion as aforesaid, shall be allocated to the QIB Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. 222 ISSUE PROCEDURE Book Building Procedure The Issue is being made through the 100% Book Building Process wherein up to 50% of the Net Issue to the public shall be available for allocation on a discretionary basis to QIB Bidders. Further not less than 35% of the Net Issue to the public shall be available for allocation on a proportionate basis to the Retail Individual Bidders and not less than 15% of the Net Issue to the public shall be available for allocation on a proportionate basis to Non-Institutional Bidders, subject to valid Bids being received at or above the Issue Price. Bidders are required to submit their Bids through the Syndicate. Our Bank in consultation with the BRLMs, reserves the right to reject any Bid procured from QIB Bidders, by any or all members of the Syndicate, without assigning any reason thereof. In case of Non-Institutional Bidders, Retail Individual Bidders, Bids under Employees Reservation Portion and Bids under Existing Shareholders Reservation Portion, our Bank would have a right to reject the Bids only on technical grounds. Investors should note that Allotment to all successful Bidders will only be in the dematerialised form. Bidders will not have the option of getting Allotment in physical form. The Equity Shares, on Allotment, shall be traded only in the dematerialised segment of the Stock Exchanges. Bid cum Application Form Bidders shall only use the specified Bid cum Application Form bearing the stamp of a member of the Syndicate for the purpose of making a Bid in terms of this Red Herring Prospectus. The Bidder shall have the option to make a maximum of three Bids in the Bid cum Application Form and such options shall not be considered as multiple Bids. Upon the allocation of Equity Shares, dispatch of the CAN, and filing of the Prospectus with the Designated Stock Exchange, the Bid cum Application Form shall be considered as the application form. Upon completing and submitting the Bid cum Application Form to a member of the Syndicate, the Bidder is deemed to have authorised our Bank to make the necessary changes in this Red Herring Prospectus and the Bid cum Application Form as would be required for filing the Prospectus with the Designated Stock Exchange and as would be required by Designated Stock Exchange after such filing, without prior or subsequent notice of such changes to the Bidder. The prescribed colour of the Bid cum Application Form for various categories, is as follows: Category Colour of Bid cum Application Form Indian public including resident QIBs, White Non-Institutional Bidders and Retail Individual Bidders Eligible NRIs and FIIs Blue Employees Pink Existing Shareholders Green Who can Bid? 1. Indian nationals resident in India who are majors, in single or joint names (not more than three); 2. Hindu Undivided Families or HUFs in the individual name of the karta. The Bidder should specify that the Bid is being made in the name of the HUF in the Bid cum Application Form as follows: “Name of Sole or First Bidder: XYZ Hindu Undivided Family applying through XYZ, where XYZ is the name of the Karta”. Bids by HUFs would be considered at par with those from individuals; 3. Insurance companies registered with the Insurance Regulatory and Development Authority; 4. As permitted by applicable law, provident funds with minimum corpus of Rs. 250 million and who are authorised under their constitution to invest in equity shares; 5. Pension funds with a minimum corpus of Rs. 250 million and who are authorised under their constitution to invest in equity shares; 223 6. Companies, corporate bodies and societies registered under the applicable laws in India and authorised to invest in the equity shares; 7. Indian mutual funds registered with SEBI; 8. Indian financial institutions, commercial banks, regional rural banks, co-operative banks (subject to RBI regulations and SEBI guidelines and regulations, as applicable); 9. Multilateral and bilateral development financial institutions; 10. State Industrial Development Corporations; 11. Trusts/ societies registered under the Societies Registration Act, 1860, as amended, or under any other law relating to trusts/societies and who are authorised under their constitution to hold and invest in the equity shares; 12. Eligible NRIs and other Non Residents including FIIs on a repatriation basis or a non-repatriation basis subject to applicable laws. NRIs other than Eligible NRIs, domestic venture capital funds and foreign venture capital funds are not permitted to participate in this Issue; and 13. Scientific and/ or industrial research organisations authorised to invest in equity shares. Note: The BRLMs, the Syndicate Members and any associate of the BRLMs and Syndicate Members (except asset management companies on behalf of mutual funds, Indian financial institutions and public sector banks) cannot participate in that portion of the Issue where allocation is discretionary and will not be eligible in the QIB Portion. Further, the BRLMs and Syndicate Members shall not be entitled to subscribe to this Issue in any manner except towards fulfilling their underwriting obligation. Bidders are advised to ensure that any single Bid from them does not exceed the investment limits or maximum number of Equity Shares that can be held by them under applicable law. As per the current regulations, the following restrictions are applicable for investments by mutual funds: No mutual fund scheme shall invest more than 10% of its net asset value in the Equity Shares or equity related instruments of any company provided that the limit of 10% shall not be applicable for investments in index funds or sector or industry specific funds. No mutual fund under all its schemes should own more than 10% of any company’s paid-up share capital carrying voting rights. These limits would have to be adhered to by the mutual funds for investment in Equity Shares of our Bank. As per the current regulations, the following restrictions are applicable for investments by FIIs: Foreign investment in a corresponding new bank is regulated by the provisions of the Bank Acquisition Act as applicable to our Bank. Under Section 3(2D) of the Bank Acquisition Act, foreign investment in a corresponding new bank is subject to an overall statutory limit of 20% of the paid up capital of the corresponding new bank. For corresponding new banks, the RBI monitors the ceilings on Non Resident investments on a daily basis. For effective monitoring the RBI has fixed cut off points lower than the actual ceiling which is 18% for public sector banks. For details see the section titled “Regulations and Policies-Restriction on Transfer of Shares” on page 176. The above information is given for the benefit of the Bidders. Our Bank and the BRLMs are not liable for any amendments or modification or changes in applicable laws or regulations, which may happen after the date of this Red Herring Prospectus. Bidders are advised to make their independent investigations and ensure that the number of Equity Shares bid for do not exceed the applicable limits under laws or regulations. Maximum and Minimum Bid Size (a) For Retail Individual Bidders: The Bid must be for a minimum of [ ] Equity Shares and in multiples of [ ] Equity Share thereafter, so as to ensure that the Bid Amount payable by the Bidder does not exceed Rs. 100,000. In case of revision of Bids, the Retail Individual Bidders have to ensure that the Bid Amount does not exceed Rs. 100,000. In case the Bid Amount is over Rs. 100,000 due to revision of the Bid or revision of the Price Band or on exercise of cut-off option, the Bid would be considered for allocation under the Non Institutional Bidders portion. The cut-off option is an option given only to the Retail Individual Bidders indicating their agreement to Bid and purchase at the final Issue Price as determined at the end of the Book Building Process. (b) For Non-Institutional Bidders and QIB Bidders: The Bid must be for a minimum of such number of Equity Shares such that the Bid Amount exceeds Rs. 100,000 and in multiples of [ ] Equity Shares thereafter. A Bid cannot be submitted for more 224 than the Net Issue to the public. However, the maximum Bid by a QIB Bidder should not exceed the investment limits prescribed for them by applicable laws. Under existing SEBI guidelines, a QIB Bidder cannot withdraw its Bid after the Bid Closing Date/Issue Closing Date. In case of revision in Bids, the Non Institutional Bidders, who are individuals, have to ensure that the Bid Amount is greater than Rs. 100,000 for being considered for allocation in the Non Institutional Portion. In case the Bid Amount reduces to Rs. 100,000 or less due to a revision in Bids or revision of the Price Band, Bids by Non Institutional Bidders who are eligible for allocation in the Retail Portion would be considered for allocation under the Retail Portion. Non Institutional Bidders and QIB Bidders are not allowed to Bid at ‘Cut-off Price’. (c) For Bidders in the Employees Reservation Portion: The Bid must be for a minimum of [ ] Equity Shares and in multiples of [ ] Equity Shares thereafter. The maximum Bid in this portion cannot exceed 500 Equity Shares. Bidders in the Employees Reservation Portion applying for a maximum Bid in any of the bidding options not exceeding Rs. 100,000 may bid at ‘Cut- off Price’. (d) For Bidders in the Existing Shareholder Reservation Portion: The Bid must be for a minimum of [ ] Equity Shares and in multiples of [ ] Equity Shares thereafter. Bidders in the Existing Shareholders Reservation Portion applying for a maximum Bid in any of the Bidding Options not exceeding Rs. 100,000 may bid at ‘Cut-off Price’. Information for the Bidders (a) Our Bank will file the Red Herring Prospectus with the Designated Stock Exchange at least three days before the Bid Opening Date/ Issue Opening Date. (b) The Price Band shall be advertised at least one day prior to the Bid Opening Date/Issue Opening Date. With regard to the Price Band, the Bidders can be guided by the secondary market prices of the Equity Shares. (c) The members of the Syndicate will circulate copies of the Red Herring Prospectus along with the Bid cum Application Form to potential investors. (d) Any investor (who is eligible to invest in our Equity Shares according to the terms of this Red Hearing Prospectus and applicable law) who would like to obtain the Red Herring Prospectus and/ or the Bid cum Application Form can obtain the same from our Head Office or from any of the members of the Syndicate. (e) The Bids should be submitted on the prescribed Bid cum Application Form only. Bid cum Application Forms should bear the stamp of the members of the Syndicate. Bid cum Application Forms which do not bear the stamp of the members of the Syndicate will be rejected. Method and Process of Bidding (a) Our Bank and the BRLMs shall declare the Bid Opening Date/ Issue Opening Date, Bid Closing Date/Issue Closing Date at the time of filing the Red Herring Prospectus with the Designated Stock Exchange and also publish the same in two widely circulated newspapers (one each in English and Hindi). This advertisement shall contain the salient features of the Red Herring Prospectus in the nature of the specifications under Form 2A of the Companies Act, the method and process of bidding and the names and addresses of the BRLMs and the Syndicate Members and their bidding centers. The Price Band shall be disclosed at least one day prior to Bid Opening Date/Issue Opening Date. The Syndicate Members shall accept Bids from the Bidders during the Issue Period in accordance with the terms of the Syndicate Agreement. (b) Investors who are interested in subscribing for the Equity Shares should approach any of the members of the Syndicate or their authorised agent(s) to register their Bid. (c) The Bidding Period shall be a minimum of three days and not exceed seven days. In case the Price Band is revised, the revised Price Band and Bidding Period will be published in two national newspapers (one each in English and Hindi) and the Bidding Period may be extended, if required, by an additional three days, subject to the total Bidding Period not exceeding 13 days. (d) Each Bid cum Application Form will give the Bidder the choice to bid for up to three optional prices (for details refer to the section titled “Issue Procedure - Bids at Different Price Levels” on page 226 below) within the Price Band and specify the demand (i.e. the number of Equity Shares Bid for) in each option. The price and demand options submitted by the Bidder in the Bid cum Application Form will be treated as optional demands from the Bidder and will not be cumulated. After 225 determination of the Issue Price, the maximum number of Equity Shares Bid for by a Bidder at or above the Issue Price will be considered for allocation and the rest of the Bid(s), irrespective of the Bid Price, will become automatically invalid. (e) The Bidder cannot bid on another Bid cum Application Form after Bids on one Bid cum Application Form have been submitted to any member of the Syndicate. Submission of a second Bid cum Application Form to either the same or to another member of the Syndicate will be treated as multiple Bids and is liable to be rejected either before entering the Bid into the electronic bidding system, or at any point of time prior to the allocation or allotment of Equity Shares in this Issue. However, the Bidder can revise the Bid through the Revision Form, the procedure for which is detailed under the section titled “Issue Procedure - Build up of the Book and Revision of Bids” on page 228. (f) The Syndicate Members will enter each Bid option into the electronic bidding system as a separate Bid and generate a Transaction Registration Slip, (“TRS”), for each price and demand option and give the same to the Bidder. Therefore, a Bidder can receive up to three TRSs for each Bid cum Application Form. (g) During the Bidding Period, Bidders may approach the members of the Syndicate to submit their Bid. Every member of the Syndicate shall accept Bids from all clients / investors who place orders through them and shall have the right to vet the Bids. (h) Along with the Bid cum Application Form, all Bidders will make payment in the manner described under the section titled “Issue Procedure - Terms of Payment and Payment into the Escrow Accounts” on page 227. Bids at Different Price Levels (a) The Price Band shall be advertised at least one day prior to the Bid Opening Date/ Issue Opening Date in Business Standard, an English language newspaper with wide circulation and Hindustan, a Hindi language newspaper with wide circulation. Bidders are advised to be guided by the price of our listed Equity Shares in the secondary market for the purposes of making a decision to invest in the Equity Shares offered as part of this Issue.The Bidders can bid at any price within the Price Band, in multiples of Re. 1. (b) In accordance with SEBI Guidelines, the Bank reserves the right to revise the Price Band during the Bidding Period. The cap on the Price Band should not be more than 20% of the floor of the Price Band. Subject to compliance with the immediately preceding sentence, the floor of the Price Band can move up or down to the extent of 20% of the floor of the Price Band disclosed in the Red Herring Prospectus. (c) In case of revision in the Price Band, the Issue Period will be extended for three additional days after revision of Price Band subject to a maximum of 13 days. Any revision in the Price Band and the revised Bidding Period/Issue Period, if applicable, will be widely disseminated by notification to BSE and NSE, by issuing a public notice in two national newspapers (one each in English and Hindi), and also by indicating the change on the websites of the BRLMs and at the terminals of the members of the Syndicate. (d) Our Bank, in consultation with the BRLMs, can finalise the Issue Price within the Price Band in accordance with this clause, without the prior approval of, or intimation, to the Bidders. (e) Bidders can bid at any price within the Price Band. Bidders have to bid for the desired number of Equity Shares at a specific price. Retail Individual Bidders, Employees and Existing Shareholders applying for a maximum Bid in any of the bidding options not exceeding Rs.100,000 may bid at Cut-off Price. However, bidding at Cut-off Price is prohibited for QIB Bidders and Non-Institutional Bidders and such Bids from QIB Bidders and Non-Institutional Bidders shall be rejected. (f) Retail Individual Bidders or Employees or Existing Shareholders who bid at the Cut-off Price agree that they shall purchase the Equity Shares at any price within the Price Band. Retail Individual Bidders or Employees or Existing Shareholders bidding at Cut-off Price shall deposit the Bid Amount based on the Cap Price in the respective Escrow Accounts. In the event the Bid Amount is higher than the subscription amount payable by the Retail Individual Bidders or Employees or Existing Shareholders, who Bid at Cut-off Price (i.e. the total number of Equity Shares allocated in the Issue multiplied by the Issue Price), the Retail Individual Bidders or Employees or Existing Shareholders, who Bid at Cut off Price, shall receive the refund of the excess amounts from the respective Escrow Accounts/ refund account(s). (g) In case of an upward revision in the Price Band announced as above, Retail Individual Bidders or Employees or Existing Shareholders, who had bid at Cut-off Price could either (i) revise their Bid or (ii) make additional payment based on the cap of the revised Price Band (such that the total amount i.e. original Bid Amount plus additional payment does not exceed Rs. 100,000 if the Bidder wants to continue to bid at Cut-off Price), with the Syndicate Member to whom the original Bid was 226 submitted. In case the total amount (i.e. original Bid Amount plus additional payment) exceeds Rs. 100,000, the Bid will be considered for allocation under the Non-Institutional Portion in terms of this Red Herring Prospectus. If, however, the Bidder does not either revise the Bid or make additional payment and the Issue Price is higher than the cap of the Price Band prior to the revision, the number of Equity Shares Bid for shall be adjusted downwards for the purpose of Allotment, such that the no additional payment would be required from the Bidder and the Bidder is deemed to have approved such revised Bid at Cut-off Price. (h) In case of a downward revision in the Price Band, announced as above, Retail Individual Bidders or Employees or Existing Shareholders, who have bid at Cut-off Price could either revise their Bid or the excess amount paid at the time of bidding would be refunded from the respective Escrow Accounts/refund account(s). (i) In the event of any revision in the Price Band, whether upwards or downwards, the minimum application size shall remain [ ] Equity Shares irrespective of whether the Bid Amount payable on such minimum application is not in the range of Rs.5,000 to Rs. 7,000. Escrow Mechanism Our Bank shall open Escrow Accounts with one or more Escrow Collection Bank(s) in whose favour the Bidders shall make out the cheque or demand draft in respect of his or her Bid and/or revision of the Bid. Cheques or demand drafts received for the full Bid Amount from Bidders in a certain category would be deposited in the respective Escrow Account. The Escrow Collection Bank(s) will act in terms of this Red Herring Prospectus and the Escrow Agreement. The monies in the Escrow Accounts shall be maintained by the Escrow Collection Bank(s) for and on behalf of the Bidders. The Escrow Collection Bank(s) shall not exercise any lien whatsoever over the monies deposited therein and shall hold the monies therein in trust for the Bidders. On the Designated Date, the Escrow Collection Bank(s) shall transfer the monies from the Escrow Accounts to the Issue Account as per the terms of the Escrow Agreement. Payments of refund to the Bidders shall also be made from the Escrow Accounts/ refund account(s) as per the terms of the Escrow Agreement and this Red Herring Prospectus. The Bidders should note that the escrow mechanism is not prescribed by SEBI and has been established as an arrangement between us, the members of the Syndicate, the Escrow Collection Bank(s) and the Registrar to the Issue to facilitate collections from the Bidders. Terms of Payment and Payment into the Escrow Accounts Each Bidder, who is required to pay Margin Amount greater than 0% shall, with the submission of the Bid cum Application Form draw a cheque or demand draft for the maximum amount of his/ her Bid in favour of the Escrow Account of the Escrow Collection Bank(s) (for details refer to the section titled “Issue Procedure - Payment Instructions” on page 235 and submit the same to the member of the Syndicate to whom the Bid is being submitted. Bid cum Application Forms accompanied by cash shall not be accepted. The maximum Bid price has to be paid at the time of submission of the Bid cum Application Form based on the highest bidding option of the Bidder. The members of the Syndicate shall deposit the cheque or demand draft with the Escrow Collection Bank(s), which will hold the monies for the benefit of the Bidders till the Designated Date. On the Designated Date, the Escrow Collection Bank(s) shall transfer the funds equivalent to the size of the Issue from the Escrow Accounts, as per the terms of the Escrow Agreement, into the Issue Account with the Banker(s) to the Issue. The balance amount after transfer to the Issue Account shall be held for the benefit of the Bidders who are entitled to refunds on the Designated Date, and not later than 15 days from the Bid Closing Date / Issue Closing Date, the Escrow Collection Bank(s) shall refund all monies to unsuccessful Bidders and also the excess amount paid on bidding, if any, after adjustment for Allotment to the Bidders. Each category of Bidders i.e. QIB Bidders, Non-Institutional Bidders, Retail Individual Bidders, Employees and Existing Shareholders would be required to pay their applicable Margin Amount at the time of the submission of the Bid cum Application Form. The Margin Amount payable by each category of Bidders is mentioned in the section titled “Issue Structure” on page 220. Where the Margin Amount applicable to the Bidder is less than 100% of the Bid Amount, any difference between the amount payable by the Bidder for Equity Shares allocated at the Issue Price and the Margin Amount paid at the time of Bidding, shall be payable by the Bidder not later than the Pay-in-Date, which shall be a minimum period of two days from the date of communication of the allocation list to the members of the Syndicate by the BRLMs. If the payment is not made favouring the appropriate Escrow Account within the time stipulated above, the Bid of the Bidder is liable to be rejected. However, if the members of the Syndicate, do not waive such payment, the full amount of payment has to be made at the time of submission of the bid form. 227 Where the Bidder has been allocated lesser number of Equity Shares than he or she had Bid for, the excess amount paid on bidding, if any, after adjustment for allocation, will be refunded to such Bidder within 15 days from the Bid Closing Date/Issue Closing Date, failing which we and shall pay interest at 15% per annum for any delay beyond the periods as mentioned above. Electronic Registration of Bids (a) The Syndicate Members will register the Bids using the on-line facilities of NSE and BSE. There will be at least one on-line connectivity in each city, where a stock exchange is located in India and where Bids are being accepted. (b) NSE and BSE will offer a screen-based facility for registering Bids for the Issue. This facility will be available on the terminals of the Syndicate Members and their authorised agents during the Bidding Period/Issue Period. Syndicate Members can also set up facilities for off-line electronic registration of Bids subject to the condition that they will subsequently download the off-line data file into the on-line facilities for book building on an half hourly basis. On the Bid Closing Date/ Issue Closing Date, the Syndicate Members shall upload the Bids till such time as may be permitted by the Stock Exchanges. (c) The aggregate demand and price for Bids registered on the electronic facilities of NSE and BSE will be downloaded on an half hourly basis, consolidated and displayed on-line at all bidding centers. A graphical representation of the consolidated demand and price would be made available at the bidding centers during the Bidding Period/ Issue Period. (d) At the time of registering each Bid, the members of the Syndicate shall enter the following details of the investor in the on- line system: Name of the investor. Investor Category – Employee, Existing Shareholder, Individual, Corporate, FII, Eligible NRI or mutual fund etc. Numbers of Equity Shares Bid for. Bid price. Bid cum Application Form number. Whether payment is made upon submission of Bid cum Application Form. Depository Participant identification no. and client identification no. of the demat account of the Bidder. (e) A system generated TRS will be given to the Bidder as a proof of the registration of each of the bidding options. It is the Bidder’s responsibility to obtain the TRS from the members of the Syndicate. The registration of the Bid by the member of the Syndicate does not guarantee that the Equity Shares shall be allocated either by the members of the Syndicate or the Bank. (f) Such TRS will be non-negotiable and by itself will not create any obligation of any kind. (g) In case of QIB Bidders, the members of the Syndicate also have the right to accept the Bid or reject it without assigning any reason. In case of Bids under the Non-Institutional Portion, Bids under the Retail Portion, Bids under the Employees Reservation Portion and Bids under Existing Shareholders Reservation Portion, Bids would not be rejected except on the technical grounds listed in this Red Herring Prospectus. (h) It is to be distinctly understood that the permission given by NSE and BSE to use their network and software of the Online IPO system should not in any way be deemed or construed to mean that the compliance with various statutory and other requirements by our Bank or the BRLMs are cleared or approved by NSE and BSE; nor does it in any manner warrant, certify or endorse the correctness or completeness of any of the compliance with the statutory and other requirements nor does it take any responsibility for the financial or other soundness of our Bank, our Promoter, our management or any scheme or project of our Bank. (i) It is also to be distinctly understood that the approval given by NSE and BSE should not in any way be deemed or construed that this Red Herring Prospectus has been cleared or approved by the NSE and BSE; nor does it in any manner warrant, certify or endorse the correctness or completeness of any of the contents of this Red Herring Prospectus; nor does it warrant that the Equity Shares will be listed or will continue to be listed on the NSE and BSE. Build Up of the Book and Revision of Bids (a) Bids registered by various Bidders through the Syndicate Members shall be electronically transmitted to the NSE or BSE mainframe on a half hourly basis. 228 (b) The book gets built up at various price levels. This information will be available with the BRLMs on a half hourly basis. (c) During the Bidding Period, any Bidder who has registered his or her interest in the Equity Shares at a particular price level is free to revise his or her Bid within the Price Band using the printed Revision Form, which is a part of the Bid cum Application Form. (d) Revisions can be made in both the desired number of Equity Shares and the Bid price by using the Revision Form. Apart from mentioning the revised options in the Revision Form, the Bidder must also mention the details of all the options in his or her Bid cum Application Form or earlier Revision Form. For example, if a Bidder has Bid for three options in the Bid cum Application Form and he is changing only one of the options in the Revision Form, he must still fill the details of the other two options that are not being changed, in the Revision Form unchanged. Incomplete or inaccurate Revision Forms will not be accepted by the members of the Syndicate. (e) The Bidder can make this revision any number of times during the Bidding Period. However, for any revision(s) in the Bid, the Bidders will have to use the services of the same member of the Syndicate through whom he or she had placed the original Bid. Bidders are advised to retain copies of the blank Revision Form and the revised Bid must be made only in such Revision Form or copies thereof. (f) Any revision of the Bid shall be accompanied by payment in the form of cheque or demand draft for the incremental amount, if any, to be paid on account of the upward revision of the Bid. The excess amount, if any, resulting from downward revision of the Bid would be returned to the Bidder at the time of refund in accordance with the terms of this Red Herring Prospectus. In case of QIB Bidders, the members of the Syndicate may at their sole discretion waive the payment requirement at the time of one or more revisions by the QIB Bidders. (g) When a Bidder revises his or her Bid, he or she shall surrender the earlier TRS and get a revised TRS from the members of the Syndicate. It is the responsibility of the Bidder to request for and obtain the revised TRS, which will act as proof of his or her having revised the previous Bid. (h) In case of discrepancy of data between NSE or BSE and the members of the Syndicate, the decision of the BRLMs, based on the physical records of Bid cum Application Forms, shall be final and binding on all concerned. Price Discovery and Allocation (a) After the Bid Closing Date /Issue Closing Date, the BRLMs will analyse the demand generated at various price levels and discuss pricing strategy with us. (b) Our Bank, in consultation with the BRLMs, shall finalise the “Issue Price”, the number of Equity Shares to be Allotted in each portion and the allocation to successful QIB Bidders. The allocation will be decided based inter alia, on the quality of the Bidder, and the size, price and time of the Bid. (c) The allocation for QIB Bidders for up to 50% of the Net Issue would be discretionary. The allocation to Non-Institutional Bidders and Retail Individual Bidders of not less than 15% and 35% of the Net Issue respectively would be on proportionate basis, in the manner specified in the SEBI Guidelines, in consultation with Designated Stock Exchange, subject to valid Bids being received at or above the Issue Price. The allocation under the Employees Reservation Portion and the Existing Shareholders Portion for up to 10% of the Issue each would be on proportionate basis, in the manner specified in the SEBI Guidelines, in consultation with Designated Stock Exchange, subject to valid Bids being received at or above the Issue Price. (d) Any undersubscription in the Employees Reservation Portion and Existing Shareholders Reservation Portion would be included in the Net Issue to the public and first be distributed equally between the Retail Portion and the Non-Institutional Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. In the event that the demand in either of the Retail Portion or the Non-Institutional Portion has been met, the Equity Shares shall be allocated to the portion in which the demand has not been met. The remaining undersubscribed Equity Shares, if any, after allocation to the Bidders in the Retail Portion and the Non-Institutional Portion as aforesaid, shall be allocated to the QIB Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. (e) Allocation to QIBs, Non Residents, FIIs and Eligible NRIs applying on repatriation basis will be subject to the terms and conditions stipulated by the RBI while granting permission for Allotment of Equity Shares to them. 229 (f) The BRLMs, in consultation with us, shall notify the members of the Syndicate of the Issue Price and allocations to their respective Bidders, where the full Bid Amount has not been collected from the Bidders. (g) We reserve the right to cancel the Issue any time after the Bid Opening Date/Issue Opening Date without assigning any reasons whatsoever. (h) In terms of SEBI Guidelines, QIB Bidders shall not be allowed to withdraw their Bid after the Bid Closing Date/ Issue Closing Date. Signing of Underwriting Agreement and Filing with the Designated Stock Exchange (a) Our Bank, the BRLMs and the Syndicate Members shall enter into an Underwriting Agreement on finalisation of the Issue Price and allocation(s) to the Bidders. (b) After signing the Underwriting Agreement, we would update and file the updated Red Herring Prospectus with the Designated Stock Exchange, which then would be termed ‘Prospectus’. The Prospectus would have details of the Issue Price, Issue size, underwriting arrangements and would be complete in all material respects. Advertisement regarding Issue Price and Prospectus A statutory advertisement will be issued by the Bank after the filing of the Prospectus with the Designated Stock Exchange. This advertisement, in addition to the information that has to be set out in the statutory advertisement, shall indicate the Issue Price. Any material updates between the date of Red Herring Prospectus and the date of Prospectus will be included in such statutory advertisement. Issuance of CAN (a) The BRLMs or Registrar to the Issue shall send to the members of the Syndicate a list of their Bidders who have been allocated Equity Shares in the Issue. (b) The BRLMs or members of the Syndicate would then send the CAN to their Bidders who have been allocated Equity Shares in the Issue. The dispatch of a CAN shall be deemed a valid, binding and irrevocable contract for the Bidder to pay the entire Issue Price for all the Equity Shares allocated to such Bidder. Those Bidders who have not paid into the Escrow Accounts at the time of bidding shall pay in full the amount payable into the Escrow Accounts by the Pay-in Date specified in the CAN. (c) Bidders who have been allocated Equity Shares and who have already paid into the Escrow Account at the time of bidding shall directly receive the CAN from the Registrar to the Issue subject, however, to realisation of their cheque or demand draft paid into the Escrow Accounts. The dispatch of a CAN shall be a deemed a valid, binding and irrevocable contract for the Bidder to pay the entire Issue Price for the Allotment to such Bidder. Designated Date and Allotment of Equity Shares (a) Our Bank will ensure that the Allotment of Equity Shares is done within 15 days of the Bid Closing Date/Issue Closing Date. After the funds are transferred from the Escrow Accounts to the Issue Account on the Designated Date, our Bank would ensure the credit to the successful Bidders depository account Allotment of the Equity Shares to the allottees within two working days of the date of Allotment. (b) As per SEBI Guidelines, Equity Shares will be issued, transferred and Allotment shall be made only in the dematerialised form to the allottees. Allottees will have the option to re-materialise the Equity Shares, if they so desire, in the manner stated in the Depositories Act. Investors are advised to instruct their Depository Participant to accept the Equity Shares that may be Allotted to them pursuant to this Issue. 230 GENERAL INSTRUCTIONS Do’s: a) Check if you are eligible to apply; b) Read all the instructions carefully and complete the Resident Bid cum Application Form (white in colour) or Non-Resident Bid cum Application Form (blue in colour), or Employee Bid Cum Application Form (pink in colour) or Existing Shareholder Bid cum Application Form (specified pre printed form, green in colour, which shall be mailed to them) as the case may be; c) Ensure that the details about Depository Participant and Beneficiary Account are correct as Allotment of Equity Shares will be in the dematerialized form only; d) Ensure that the Bids are submitted at the bidding centers only on forms bearing the stamp of a member of the Syndicate; e) Ensure that you have been given a TRS for all your Bid options; f) Submit revised Bids to the same member of the Syndicate through whom the original Bid was placed and obtain a revised TRS; g) Ensure that you mention your Permanent Account Number (PAN) allotted under the I.T. Act where the maximum Bid for Equity Shares by a Bidder is for a total value of Rs. 50,000 or more. In case the PAN has not been allotted, mention “Not allotted” in the appropriate place; and h) Ensure that the Demographic Details (as defined hereinbelow) are updated, true and correct in all respects. Don’ts: (a) Do not Bid for lower than the minimum Bid size; (b) Do not Bid/ revise Bid price to less than the lower end of the price band or higher than the higher end of the Price Band; (c) Do not Bid on another Bid cum Application Form after you have submitted a Bid to the members of the Syndicate; (d) Do not pay the Bid Amount in cash; (e) Do not send Bid cum Application Forms by post; instead submit the same to a member of the Syndicate only; (f) Do not Bid at Cut-off Price (for QIB Bidders, Non-Institutional Bidders, Employees and Existing Shareholders for whom the Bid Amount exceeds Rs 100,000); (g) Do not fill up the Bid cum Application Form such that the Equity Shares Bid for exceeds the Net Issue size and/ or investment limit or maximum number of Equity Shares that can be held under the applicable laws or regulations or maximum amount permissible under the applicable regulations; (h) Do not submit Bid accompanied with Stockinvest. (i) Do not provide your GIR number instead of your PAN. INSTRUCTIONS FOR COMPLETING THE BID CUM APPLICATION FORM Bidders can obtain Bid cum Application Forms and / or Revision Forms from the members of the Syndicate. Bids and Revisions of Bids Bids and revisions of Bids must be: (a) Made only in the prescribed Bid cum Application Form or Revision Form, as applicable (white colour for Resident Indians, blue colour for Eligible NRIs and FIIs and applying on repatriation basis, pink colour for Employees and the specified pre printed form for Existing Shareholders which shall be mailed to them). (b) Completed in full, in BLOCK LETTERS in ENGLISH and in accordance with the instructions contained herein, in the Bid cum Application Form or in the Revision Form. Incomplete Bid cum Application Forms or Revision Forms are liable to be rejected. (c) The Bids from the Retail Individual Bidders must be for a minimum of [ ] Equity Shares and in multiples of [ ] thereafter subject to a maximum Bid Amount of Rs. 100,000. 231 (d) For Non-institutional Bidders and QIB Bidders, Bids must be for a minimum of such number of Equity Shares that the Bid Amount exceeds Rs 100,000 and in multiples of [ ] Equity Shares thereafter. Bids cannot be made for more than the Net Issue size. Bidders are advised to ensure that a single Bid from them should not exceed the investment limits or maximum number of Equity Shares that can be held by them under the applicable laws or regulations. (e) For Employees, the Bid must be for a minimum of [ ] Equity Shares and shall be in multiples of [ ] Equity Shares thereafter. The maximum Bid Amount in this portionis 500 Equity Shares. (f) For Existing Shareholders, the Bid must be for a minimum of [ ] Equity Shares and shall be in multiples of [ ] Equity Shares thereafter. (g) In single name or in joint names (not more than three, and in the same order as their Depository Participant details). (h) Thumb impressions and signatures other than in the languages specified in the Eighth Schedule of the Constitution of India must be attested by a Magistrate or a Notary Public or a Special Executive Magistrate under official seal. Bids by Employees For the sake of clarity, the term “Employees” shall mean all or any of the following: (a) a permanent employee of the Bank working in India as of January 1, 2005; (b) a director of the Bank, whether a whole time director, part time director or otherwise as of January 1, 2005. 1. Bids by Employees shall be made only in the prescribed Bid cum Application Form or Revision Form, (i.e. pink colour form). 2. Employees should mention their provident fund number at the relevant place in the Bid cum Application Form. 3. The sole/First Bidder should be an Employee. In case the Bid cum Application Form is submitted in joint names, it should be ensured that the Depository Account is also held in the same joint names and in the same sequence in which they appear in the Bid cum Application Form. 4. Only Employees on the rolls of the Bank as on January 1, 2005 would be eligible to apply in this Issue under reservation for Employees on a competitive basis. 5. Employees will have to Bid like any other Bidder. Only those Bids, which are received at or above the Issue Price, would be considered for allotment under the Employees Reservation Portion. 6. The maximum Bid in this portion can be for 500 Equity Shares. 7. If the aggregate demand in this category is less than or equal to 8,000,000 Equity Shares at or above the Issue Price, full allocation shall be made to the Employees to the extent of their demand. Any undersubscription in the Employees Reservation Portion would be included in the Net Issue to the public and first be distributed equally between the Retail Portion and the Non-Institutional Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. In the event that the demand in either of the Retail Portion or the Non- Institutional Portion has been met, the Equity Shares shall be allocated to the portion in which the demand has not been met. The remaining undersubscribed Equity Shares, if any, after allocation to the Bidders in the Retail Portion and the Non- Institutional Portion as aforesaid, shall be allocated to the QIB Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. 8. If the aggregate demand in this portion is greater than 8,000,000 Equity Shares at or above the Issue Price, the allocation shall be made on a proportionate basis subject to a minimum of [ ] Equity Shares. For the method of proportionate basis of allocation, refer to section titled “Statutory and other Information - Basis of Allocation” on page 244. 9. Bidding at Cut-off is allowed only for Employees whose Bid Amount is less than or equal to Rs 100,000. Bids by Existing Shareholders For the sake of clarity, the term “Existing Shareholders” shall mean the natural persons, who are holders of Equity Shares of the Bank as of February 11, 2005 and who hold Equity Shares worth up to Rs. 100,000 determined on the basis of closing price of the Equity Shares in the NSE on February 10, 2005. 1. Bids by Existing Shareholders shall be made only in the prescribed Bid cum Application Form or Revision Form, which shall be mailed to them. 232 2. Existing Shareholders should mention their Registered Folio Number/DP and Client ID number at the relevant place in the Bid cum Application Form. 3. The sole/First Bidder should be an Existing Shareholder. In case the Bid cum Application Form is submitted in joint names, it should be ensured that the Depository Account is also held in the same joint names and in the same sequence in which they appear in the Bid cum Application Form. 4. Only Existing Shareholders of the Bank as on February 11, 2005 would be eligible to apply in this Issue under reservation for Existing Shareholders on a competitive basis. 5. Existing Shareholders will have to Bid like any other Bidder. Only those Bids, which are received at or above the Issue Price, would be considered for allotment under the Existing Shareholders Reservation Portion. 6. If the aggregate demand in this category is less than or equal to 8,000,000 Equity Shares at or above the Issue Price, full allocation shall be made to the Existing Shareholders to the extent of their demand. Any undersubscription in the Existing Shareholders Reservation Portion would be included in the Net Issue to the public and first be distributed equally between the Retail Portion and the Non-Institutional Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. In the event that the demand in either of the Retail Portion or the Non-Institutional Portion has been met, the Equity Shares shall be allocated to the portion in which the demand has not been met. The remaining undersubscribed Equity Shares, if any, after allocation to the Bidders in the Retail Portion and the Non-Institutional Portion as aforesaid, shall be allocated to the QIB Portion in accordance with the description in section titled “Statutory and other Information - Basis of Allocation” as described in page 244. 7. If the aggregate demand in this category is greater than 8,000,000 Equity Shares at or above the Issue Price, the allocation shall be made on a proportionate basis subject to a minimum of [·] Equity Shares. For the method of proportionate basis of allocation, refer to section titled “Statutory and other Information - Basis of Allocation” on page 244. 8. Bidding at Cut-off Price is allowed only for Existing Shareholders whose Bid Amount is less than or equal to Rs 100,000. Bidder’s Bank Details Bidders should note that on the basis of name of the Bidders, Depository Participant’s name, Depository Participant-Identification number and Beneficiary Account Number provided by them in the Bid cum Application Form, the Registrar to the Issue will obtain from the Depository the Bidders bank account details. These bank account details would be printed on the refund order, if any, to be sent to Bidders. Hence, Bidders are advised to immediately update their bank account details as appearing on the records of the depository participant. Please note that failure to do so could result in delays in credit of refunds to Bidders at the Bidders sole risk and neither the BRLMs nor the Bank shall have any responsibility and undertake any liability for the same. Bidder’s Depository Account Details IT IS MANDATORY FOR ALL THE BIDDERS TO GET THEIR EQUITY SHARES IN DEMATERIALISED FORM. ALL BIDDERS SHOULD MENTION THEIR DEPOSITORY PARTICIPANT’S NAME, DEPOSITORY PARTICIPANT IDENTIFICATION NUMBER AND BENEFICIARY ACCOUNT NUMBER IN THE BID CUM APPLICATION FORM. INVESTORS MUST ENSURE THAT THE NAME GIVEN IN THE BID CUM APPLICATION FORM IS EXACTLY THE SAME AS THE NAME IN WHICH THE DEPOSITORY ACCOUNT IS HELD. IN CASE THE BID CUM APPLICATION FORM IS SUBMITTED IN JOINT NAMES, IT SHOULD BE ENSURED THAT THE DEPOSITORY ACCOUNT IS ALSO HELD IN THE SAME JOINT NAMES AND ARE IN THE SAME SEQUENCE IN WHICH THEY APPEAR IN THE BID CUM APPLICATION FORM. Bidders should note that on the basis of name of the Bidders, Depository Participant’s name, Depository Participant- Identification number and Beneficiary Account Number provided by them in the Bid cum Application Form, the Registrar to the Issue will obtain from the Depository demographic details of the Bidders such as address, bank account details for printing on refund orders and occupation (“Demographic Details”). Hence, Bidders should carefully fill in their Depository Account details in the Bid cum Application Form. These Demographic Details would be used for all correspondence with the Bidders including mailing of the refund orders/ CANs/Allocation Advice and printing of bank particulars on the refund order and the Demographic Details given by Bidders in the Bid cum Application Form would not be used for these purposes by the Registrar. 233 Hence, Bidders are advised to update their Demographic Details as provided to their Depository Participants and ensure that they are true and correct. By signing the Bid cum Application Form, Bidder would have deemed to authorise the depositories to provide, upon request, to the Registrar to the Issue, the required Demographic Details as available on its records. Refund Orders/Allocation Advice/CANs would be mailed at the address of the Bidder as per the Demographic Details received from the Depositories. Bidders may note that delivery of refund orders/allocation advice/CANs may get delayed if the same once sent to the address obtained from the Depositories are returned undelivered. In such an event, the address and other details given by the Bidder in the Bid cum Application Form would be used only to ensure dispatch of refund orders. Please note that any such delay shall be at the Bidders sole risk and neither the Bank nor the BRLMs shall be liable to compensate the Bidder for any losses caused to the Bidder due to any such delay or liable to pay any interest for such delay. In case no corresponding record is available with the Depositories that matches three parameters, namely, names of the Bidders (including the order of names of joint holders), the Depository Participant’s identity (DP ID) and the beneficiary’s identity, then such Bids are liable to be rejected. Bids under Power of Attorney In case of Bids made pursuant to a power of attorney or by limited companies, corporate bodies, registered societies, a certified copy of the power of attorney or the relevant resolution or authority, as the case may be, along with a certified copy of the memorandum and articles of association and/or bye laws must be lodged along with the Bid cum Application Form. Failing this, the Bank reserves the right to accept or reject any Bid in whole or in part, in either case, without assigning any reason therefor. In case of Bids made pursuant to a power of attorney by FIIs, a certified copy of the power of attorney or the relevant resolution or authority, as the case may be, along with a certified copy of their SEBI registration certificate must be lodged along with the Bid cum Application Form. Failing this, our Bank reserves the right to accept or reject any Bid in whole or in part, in either case, without assigning any reason therefor. In case of Bids made by insurance companies registered with the Insurance Regulatory and Development Authority, a certified copy of certificate of registration issued by Insurance Regulatory and Development Authority must be lodged along with the Bid cum Application Form. Failing this, our Bank reserves the right to accept or reject any Bid in whole or in part, in either case, without assigning any reason therefor. In case of Bids made, subject to applicable law, by provident funds with minimum corpus of Rs. 250 million and pension funds with minimum corpus of Rs. 250 million, a certified copy of certificate from a chartered accountant certifying the corpus of the provident fund/ pension fund must be lodged along with the Bid cum Application Form. Failing this, our Bank reserves the right to accept or reject any Bid in whole or in part, in either case, without assigning any reason therefor. We, in our absolute discretion, reserve the right to relax the above condition of simultaneous lodging of the power of attorney along with the Bid cum Application Form, subject to such terms and conditions that we and the BRLMs may deem fit. Bids by Eligible NRIs and FIIs on a repatriation basis Bids and revision to Bids must be made: 1. On the Bid cum Application Form or the Revision Form, as applicable (blue in color), and completed in full in BLOCK LETTERS in ENGLISH in accordance with the instructions contained therein. 2. In a single name or joint names (not more than three). 3. By FIIs for a minimum of such number of Equity Shares and in multiples of [ ] thereafter that the Bid Amount exceeds Rs. 100,000. For further details see section titled “Issue Procedure - Maximum and Minimum Bid Size” on page 224. 4. In the names of individuals, or in the names of FIIs but not in the names of minors, OCBs, firms or partnerships, foreign nationals (including Eligible NRIs) or their nominees, foreign venture capital investors. 5. Refunds, dividends and other distributions, if any, will be payable in Indian Rupees only and net of bank charges and/or commission. In case of Bidders who remit money through Indian Rupee drafts purchased abroad, such payments in Indian 234 Rupees will be converted into U.S. Dollars or any other freely convertible currency as may be permitted by the RBI at the rate of exchange prevailing at the time of remittance and will be dispatched by registered post or if the Bidders so desire, will be credited to their NRE accounts, details of which should be furnished in the space provided for this purpose in the Bid cum Application Form. We will not be responsible for loss, if any, incurred by the Bidder on account of conversion of foreign currency. It is to be distinctly understood that there is no reservation for Eligible NRIs and FIIs. All Eligible NRIs and FIIs will be treated on the same basis with other categories for the purpose of allocation. PAYMENT INSTRUCTIONS We shall open Escrow Accounts with the Escrow Collection Bank(s) for the collection of the Bid Amounts payable upon submission of the Bid cum Application Form and for amounts payable pursuant to allocation in the Issue. Each Bidder shall draw a cheque or demand draft for the amount payable on the Bid and/or on allocation as per the following terms: PAYMENT INTO ESCROW ACCOUNT (i) The Bidders for whom the applicable Margin Amount is equal to 100% shall, with the submission of the Bid cum Application Form draw a payment instrument for the Bid Amount in favour of the Escrow Account and submit the same to the members of the Syndicate. (ii) In case the above Margin Amount paid by the Bidders during the Bidding Period is less than the Issue Price multiplied by the Equity Shares allocated to the Bidder, the balance amount shall be paid by the Bidders into the Escrow Account within the period specified in the CAN which shall be subject to a minimum period of two days from the date of communication of the allocation list to the members of the Syndicate by the BRLMs. (iii) The payment instruments for payment into the Escrow Account should be drawn in favour of: (a) In case of Resident Bidders: “Escrow Account –PNB FPO”; (b) In case of Non Resident Bidders: “Escrow Account –PNB FPO–NR”; (c) In case of Employees: “Escrow Account –PNB FPO–Employees”; and (d) In case of Existing Shareholders: “Escrow Account–PNB FPO–Existing Shareholders” In case of bids by Eligible NRIs applying on a repatriation basis, the payments must be made through Indian rupee drafts purchased abroad or cheques or bank drafts, for the amount payable on application remitted through normal banking channels or out of funds held in the NRE Accounts or the FCNR Accounts, maintained with banks authorised to deal in foreign exchange in India, along with documentary evidence in support of the remittance. Payment will not be accepted out of Non Resident Ordinary (NRO) Account of the Non Resident Bidder bidding on a repatriation basis. Payment by drafts should be accompanied by bank certificate confirming that the draft has been issued by debiting to the NRE Account or the FCNR Account. In case of Bids by FIIs, the payment should be made out of funds held in Special Rupee Account along with documentary evidence in support of the remittance. Payment by drafts should be accompanied by bank certificate confirming that the draft has been issued by debiting to Special Rupee Account. (iv) Where a Bidder has been allocated a lesser number of Equity Shares than the Bidder has Bid for, the excess amount, if any, paid on bidding, after adjustment towards the balance amount payable on the Equity Shares allocated, will be refunded to the Bidder from the Escrow Accounts. (v) The monies deposited in the Escrow Account will be held for the benefit of the Bidders till the Designated Date. (vi) On the Designated Date, the Escrow Collection Banks shall transfer the funds from the Escrow Account as per the terms of the Escrow Agreement into the Issue Account with the Banker to the Issue. (vii)On the Designated Date and no later than 15 days from the Bid Closing Date/Issue Closing Date, the Escrow Collection Banks shall also refund all amounts payable to unsuccessful Bidders and also the excess amount paid on Bidding, if any, 235 after adjusting for allocation to the Bidders Payments should be made by cheque, or demand draft drawn on any bank (including a Co-operative bank), which is situated at, and is a member of or sub-member of the bankers’ clearing house located at the centre where the Bid cum Application Form is submitted. Outstation cheques/bank drafts drawn on banks not participating in the clearing process will not be accepted and applications accompanied by such cheques or bank drafts are liable to be rejected. Cash/ stockinvest/money orders/ postal orders will not be accepted. Payment by Stockinvest In terms of the Reserve Bank of India Circular No. DBOD No. FSC BC 42/24.47.00/2003-04 dated November 5, 2003, the option to use the stockinvest instrument in lieu of cheques or bank drafts for payment of Bid money has been withdrawn. Hence, payment through Stockinvest would not be accepted in this Issue. SUBMISSION OF BID CUM APPLICATION FORM All Bid cum Application Forms or Revision Forms duly completed and accompanied by account payee cheques or drafts shall be submitted to the members of the Syndicate at the time of submission of the Bid. Each member of the Syndicate may, at its sole discretion, waive the requirement of payment at the time of submission of the Bid cum Application Form and Revision Form. No separate receipts shall be issued for the money payable on the submission of Bid cum Application Form or Revision Form. However, the collection center of the members of the Syndicate will acknowledge the receipt of the Bid cum Application Forms or Revision Forms by stamping and returning to the Bidder the acknowledgement slip. This acknowledgement slip will serve as the duplicate of the Bid cum Application Form for the records of the Bidder. OTHER INSTRUCTIONS Joint Bids in the case of Individuals Bids may be made in single or joint names (not more than three). In the case of joint Bids, all payments will be made out in favour of the Bidder whose name appears first in the Bid cum Application Form or Revision Form. All communications will be addressed to the First Bidder and will be dispatched to his or her address. Multiple Bids A Bidder should submit only one Bid (and not more than one) for the total number of Equity Shares required. Two or more Bids will be deemed to be multiple Bids if the sole or First Bidder is one and the same. In case of a mutual fund, a separate Bid can be made in respect of each scheme of the mutual fund registered with SEBI and such Bids in respect of more than one scheme of the mutual fund will not be treated as multiple Bids provided that the Bids clearly indicate the scheme concerned for which the Bid has been made. Bids made by Employees both under Employees Reservation Portion as well as in the Net Issue shall not be treated as multiple Bids. Bids made by Existing Shareholders both under Existing Shareholders Reservation Portion as well as in the Net Issue shall not be treated as multiple Bids. We reserve the right to reject, in our absolute discretion, all or any multiple Bids in any or all portion. Permanent Account Number or PAN Where Bid(s) is/are for Rs. 50,000 or more, the Bidder or in the case of an Bid in joint names, each of the Bidders, should mention his/her Permanent Account Number (PAN) allotted under the I.T. Act. The copy of the PAN card or PAN allotment letter is required to be submitted with the application form. Applications without this information and documents will be considered incomplete and are liable to be rejected. It is to be specifically noted that Bidders should not submit the GIR number instead of the PAN as the Bid is liable to be rejected on this ground. In case the Sole/First Bidder and Joint Bidder(s) is/are not required to obtain PAN, each of the Bidder(s) shall mention “Not Applicable” and in the event that the sole Bidder and/or the joint Bidder(s) have applied for PAN which has not yet been allotted each of the Bidder(s) should mention “Applied for” in the Bid 236 cum Application Form. Further, where the Bidder(s) has mentioned “Applied for” or “Not Applicable”, the Sole/First Bidder and each of the Joint Bidder(s), as the case may be, would be required to submit Form 60 (Form of declaration to be filed by a person who does not have a permanent account number and who enters into any transaction specified in rule 114B), or, Form 61 (form of declaration to be filed by a person who has agricultural income and is not in receipt of any other income chargeable to income- tax in respect of transactions specified in rule 114B), as may be applicable, duly filled along with a copy of any one of the following documents in support of the address: (a) Ration Card (b) Passport (c) Driving License (d) Identity Card issued by any institution (e) Copy of the electricity bill or telephone bill showing residential address (f) Any document or communication issued by any authority of the Central Government, State Government or local bodies showing residential address (g) Any other documentary evidence in support of address given in the declaration. It may be noted that Form 60 and Form 61 have been amended vide a notification issued on December 1, 2004 by the Ministry of Finance, Department of Revenue, Central Board of Direct Taxes. All Bidders are requested to furnish, where applicable, the revised Form 60 or 61, as the case may be. Unique Identification Number (“UIN”) In terms of SEBI (Central Database of Market Participants) Regulations, 2003 as amended from time to time and SEBI Notification dated November 25, 2003 and July 30, 2004, circular dated August 16, 2004 and press release dated December 31, 2004, no specified investor being a body corporate shall subscribe to securities which are proposed to be listed in any recognized stock exchange unless such specified investor, its promoters and directors have been allotted unique identification numbers (UIN) save and except: (i) those promoters or directors who are persons resident outside India, who are required to obtain UIN before December 31, 2005; and (ii) where such specified investor being a body corporate has applied for allotment of a U IN before December 31, 2004 and has not yet been allotted the UIN until the disposal of his application or, where he has filed an appeal, till the disposal of the appeal, as the case may be. In terms of the above it shall be compulsory for specified investor being a body corporate making application in this Issue to give their UIN. In case where a body corporate has made an application for such number before December 31, 2004 but the same has not been allotted, or where an appeal has been filed, but not disposed off, the investor shall indicate the same in the space provided in the Application form. Application forms from specified investors being body corporate not providing their UIN or UIN application status in ca ses which have applied for such UIN before December 31, 2004, shall be liable to be rejected. OUR RIGHT TO REJECT BIDS We and the BRLMs reserve the right to reject any Bid without assigning any reason therefor in case of QIB Bidders. In case of Non-Institutional Bidders, Retail Individual Bidders, Employees and Existing Shareholders who Bid, the Bank has a right to reject Bids on technical grounds. Consequent refunds shall be made by cheque or pay order or draft and will be sent to the Bidder’s address at the Bidder’s risk. GROUNDS FOR TECHNICAL REJECTIONS Bidders are advised to note that Bids are liable to be rejected on among others on the following technical grounds: 1. Amount paid does not tally with the amount payable for the highest value of Equity Shares Bid for; 2. Age of First Bidder not given; 3. In case of partnership firms, shares may be registered in the names of the individual partners and no firm as such, shall be entitled to apply; 4. NRIs, except Eligible NRIs, and Foreign Venture Capital Investors and Indian Venture Capital Funds; 5. Bids by Persons not competent to contract under the Indian Contract Act, 1872, including minors, insane Persons; 6. PAN Number not given if Bid is for Rs. 50,000 or more and GIR number given instead of PAN number; 7. Bids for lower number of Equity Shares than specified for that category of investors; 8. Bids at a price less than lower end of the Price Band; 9. Bids at a price more than the higher end of the Price Band; 237 10. Bids at Cut-off Price by Non-Institutional Bidders and QIB Bidders and Bids at Cut-off Price by Employees and Existing Shareholders whose Bid Amount is more than Rs. 100,000; 11. Bids for number of Equity Shares, which are not in multiples of [●]; 12. Category not ticked; 13. Multiple Bids as defined in this Red Herring Prospectus; 14. In case of Bid under power of attorney or by limited companies, corporate, trust etc., relevant documents are not submitted; 15. Bids accompanied by Stockinvest/money order/postal order/cash; 16. Signature of sole and / or joint Bidders missing; 17. Bid cum Application Forms does not have the stamp of the BRLMs or the Syndicate Members; 18. Bid cum Application Forms does not have Bidder’s depository account details; 19. Bid cum Application Forms are not delivered by the Bidders within the time prescribed as per the Bid cum Application Forms, Bid Opening Date/Issue Opening Date advertisement and this Red Herring Prospectus and as per the instructions in this Red Herring Prospectus and the Bid cum Application Forms; 20. In case no corresponding record is available with the Depositories that matches three parameters namely, names of the Bidders (including the order of names of joint holders), the Depositary Participant’s identity (DP ID) and the beneficiary’s identity; 21. Bids for amounts greater than the maximum permissible amounts prescribed by the regulations. See the details regarding the same in the section titled “Issue Procedure – Bids at Different Price Levels” at page 226; 22. Bids by OCBs; 23. Bids by US Persons other than “qualified institutional buyers” as defined in Rule 144A of the Securities Act; 24. Bids under Employees Reservation Portion for more than 500 Equity Shares. 25. Bids by specified investors being body corporates who do not provide their UIN or UIN application status, in cases which have applied for such UIN before December 31, 2004. EQUITY SHARES IN DEMATERIALISED FORM WITH NSDL OR CDSL The Allotment of Equity Shares in this Issue shall be only in a de-materialised form, (i.e. not in the form of physical certificates but be fungible and be represented by the statement issued through the electronic mode). In this context, two agreements have been signed among our Bank, the respective Depositories and the Registrar to the Issue: a) a tripartite agreement dated November 13, 2001 with NSDL, us and Registrar to the Issue in respect of debt instruments which was extended to cover equity shares vide letter dated February 26, 2002 bearing reference number AS/PM/02/393; b) a tripartite agreement dated February 25, 2002 with CDSL, us and Registrar to the Issue. All Bidders can seek Allotment only in dematerialised mode. Bids from any Bidder without relevant details of his or her depository account are liable to be rejected. a) A Bidder applying for Equity Shares must have at least one beneficiary account with either of the Depository Participants of either NSDL or CDSL prior to making the Bid. b) The Bidder must necessarily fill in the details (including the Beneficiary Account Number and Depository Participant’s identification number) appearing in the Bid cum Application Form or Revision Form. c) Allotment to a successful Bidder will be credited in electronic form directly to the beneficiary account (with the Depository Participant) of the Bidder d) Names in the Bid cum Application Form or Revision Form should be identical to those appearing in the account details in the Depository. In case of joint holders, the names should necessarily be in the same sequence as they appear in the account details in the Depository. e) If incomplete or incorrect details are given under the heading ‘Bidders Depository Account Details’ in the Bid cum Application Form or Revision Form, it is liable to be rejected. 238 f) The Bidder is responsible for the correctness of his or her demographic details given in the Bid cum Application Form vis- à-vis those with his or her Depository Participant. g) It may be noted that Equity Shares in electronic form can be traded only on the stock exchanges having electronic connectivity with NSDL and CDSL. All the Stock Exchanges where our Equity Shares are proposed to be listed have electronic connectivity with CDSL and NSDL. h) The trading of the Equity Shares of the Bank would be in dematerialised form only for all investors in the demat segment of the respective Stock Exchanges. COMMUNICATIONS All future communications in connection with Bids made in this Issue should be addressed to the Registrar to the Issue quoting the full name of the sole or First Bidder, Bid cum Application Form number, details of Depository Participant, number of Equity Shares applied for, date of Bid form, name and address of the member of the Syndicate where the Bid was submitted and cheque or draft number and issuing bank thereof. UNDERTAKINGS BY THE BANK We undertake as follows: that the complaints received in respect of this Issue shall be attended to by us expeditiously and satisfactorily; that all steps will be taken for the completion of the necessary formalities for listing and commencement of trading at all the Stock Exchanges where the Equity Shares are proposed to be listed within seven working days of finalisation of the basis of allocation; that the funds required for dispatch of refund orders or allotment advice by registered post or speed post shall be made available to the Registrar to the Issue by us; that the refund orders or allotment advice to the successful Bidders shall be dispatched within specified time; and that no further issue of Equity Shares shall be made till the Equity Shares offered through the Red Herring Prospectus are listed or until the Bid monies are refunded on account of non-listing, under-subscription etc. Our Bank shall not have recourse to the Issue proceeds until the approval for trading of the Equity Shares from all the Stock Exchanges where listing is sought has been received. DISPOSAL OF APPLICATIONS AND APPLICATIONS MONEY We shall ensure dispatch of Allotment advice, refund orders and give benefit to the beneficiary account with Depository Participants and submit the documents pertaining to the allotment to the Stock Exchanges within two working days of date of finalisation of Allotment of Equity Shares. We shall dispatch refund orders, if any, of value up to Rs. 1,500, “Under Certificate of Posting”, and shall dispatch refund orders above Rs. 1,500, if any, by registered post or speed post at the sole or First Bidder’s sole risk. We shall use best efforts to ensure that all steps for completion of the necessary formalities for listing and commencement of trading at the Stock Exchanges where the Equity Shares are proposed to be listed, are taken within seven working days of finalisation of the basis of Allotment. In accordance with the requirements of the Stock Exchanges and SEBI Guidelines, we further undertake that: Allotment shall be made only in dematerialised form within 15 days of the Bid Closing Date/Issue Closing Date; dispatch refund orders within 15 days of the Bid Closing Date/Issue Closing Date would be ensured; and we shall pay interest at 15% per annum (for any delay beyond the 15 day time period as mentioned above), if Allotment is not made and refund orders are not dispatched and/or demat credits are not made to investors within the 15 day time 239 prescribed above as per the guidelines issued by the Government of India, MoF pursuant to their letter No. F/8/S/79 dated July 31, 1983, as amended by their letter No. F/14/SE/85 dated September 27, 1985, addressed to the Stock Exchanges, and as further modified by SEBI’s Clarification XXI dated October 27, 1997, with respect to the SEBI Guidelines. Refunds will be made by cheques, pay orders or demand drafts drawn on the Escrow Collection Bank(s) and payable at par at places where Bids are received. Bank charges, if any, for cashing such cheques, pay orders or demand drafts at other centers will be payable by the Bidders. RESTRICTIONS ON FOREIGN OWNERSHIP OF INDIAN SECURITIES Foreign investment in corresponding new banks is regulated by the provisions of the Bank Acquisition Act. Under Section 3(2D) of these statutes, foreign investment in corresponding new banks is subject to an overall statutory limit of 20% of the paid up capital of the bank. Section 3(2D) Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 states as follows: “(2D) The shares of every corresponding new bank not held by the Central Government shall be freely transferable: Provided that no individual or company resident outside India or any company incorporated under any law not in force in India of any branch of such company, whether resident outside India or not, shall at any time hold or acquire by transfer or otherwise shares of the corresponding new bank so that such investment in aggregate exceed the percentage, not being more than twenty per cent of the paid-up capital as may be specified by the Central Government by notification in the Official Gazette. Explanation— For the purposes of this clause “company” means any body corporate and includes a firm or other association of individuals.” Hence, Section 3(2D) of the Bank Acquisition Act prescribed that foreign investment in the aggregate is permitted in the correspondent new bank, like our Bank only till 20% under the automatic route. For public sector banks the RBI monitors the ceilings on FII/NRI/PIO investments on a daily basis. For effective monitoring the RBI has fixed cut off points lower than the actual ceilings, which is 18% for public sector banks. For details see the section titled “Regulations and Policies-Restriction on Transfer of Shares” on page 176. 240 BASIS FOR ISSUE PRICE Qualitative Factors 1. We are India’s third largest bank in terms of assets and second largest bank in terms of number of branches. 2. We have a pan-India franchise with 4,034 branches, 452 extension counters and 467 ATMs in over 150 cities throughout India servicing 35 million customers. 3. We have a diversified loan portfolio of Rs. 472.2 billion with the corporate and commercial, retail and agriculture sectors constituting 60.8%, 19.4% and 19.8% of our total outstanding loans as on March 31, 2004. 4. Our ratio of net NPAs to net advances declined to 0.98% in fiscal 2004 from 3.86% in fiscal 2003. 5. In fiscal 2004 our interest free demand deposits and low interest savings bank deposits constituted 45% of our total deposits. These low-cost deposits led to an average cost of deposits of 5.0% and an average cost of funds of 5.1% in fiscal 2004. 6. Our deposits increased by 16 % to Rs. 879.16 billion at March 31, 2004 from Rs 758.13 billion as of March 31, 2003. 7. We have had a consistent profitability track record. Our unadjusted net profit in fiscal 2004 increased by 31.6% to Rs. 11.09 billion as compared to Rs. 8.4 billion in fiscal 2003. 8. We have 694 electronically interconnected branches as of September 30, 2004 throughout the country. Quantitative Factors Information presented in this section is derived from our unconsolidated audited restated financial statements prepared in accordance with Indian GAAP. 1. Weighted average earnings per share (EPS)* EPS Weight Year ended March 31, 2002 26.5 1 Year ended March 31, 2003 30.1 2 Year ended March 31, 2004 40.0 3 Weighted Average 34.4 The weighted average number of Equity Shares have been considered for calculation of EPS. 2. Price/Earning (P/E) ratio in relation to Issue Price of Rs. [ ]. a. Based on twelve months ended March 31, 2004 (annualised) is[ ]. b. Industry P/E(1) i) Highest: 13.9 ii) Lowest: 3.7 iii) Average (composite): 7.0 (1) Source: “Capital Market” Volume XIX/ 22 dated January 3, 2005 to January 16, 2005 for the Category titled ‘Banking – Public Sector’. The figures are in respect of fiscal 2004. 3. Weighted average return on average net worth (2) Sr. No. Year ended March 31, Return on Average Net Worth (%) Weight 1. 2002 22.10 1 2. 2003 25.12 2 3. 2004 26.86 3 Weighted Average 25.48 (2) Networth has been computed by aggregating share capital, reserves and surplus and adjusting for revaluation reserves, intangible assets and deferred tax assets as per our audited restated financial statements. 241 4. Minimum Return on Increased Net Worth Required to Maintain Pre-Issue EPS. The minimum return on increased net worth required to maintain pre-Issue EPS is [ ] to [ ]. 5. Net Asset Value per Equity Share at March 31, 2004 is Rs. 165.25. 6. Net Asset Value per Equity Share after Issue The net asset value per Equity Share after the Issue is [ ] Issue Price per Equity Share: Rs. [ ] Issue Price per Equity Share will be determined on conclusion of book building process. 7. Comparison of Accounting Ratios for year-end fiscal 2004 EPS P/E Return On Book Value Average Net Per Share Worth Peer Group (public and private sector) State Bank of India 68.5 8.2 19.7 384.4 Punjab National Bank 40.0 7.8 26.9 165.3 Bank of Baroda 31.8 7.0 20.3 173.4 Bank of India 20.5 5.8 28.0 78.5 Canara Bank 32.0 5.6 29.2 125.1 Union Bank 15.0 6.1 30.5 56.6 ICICI Bank 21.3 15.6 21.9 110.5 HDFC Bank 17.3 25.0 20.6 94.0 Peer Group (Simple) Average 30.80 10.14 24.63 148.47 Source: Our EPS, Return On Average Net Worth and Book Value Per Share have been calculated from our audited restated financial statements. Source for other information is “Capital Market” Volume XIX/ 22 dated January 3, 2005 to January 16, 2005. The Issue Price of Rs [ ] has been determined on the basis of the demand from investors through the Book-building Process and is justified based on the above accounting ratios. The face value of the Equity Shares is Rs 10 each and the Issue Price is [ ] times of the face value. 242 STATUTORY AND OTHER INFORMATION Consents Consents in writing of: (a) the Directors, the Company Secretary and Compliance Officer, the Auditors, Legal Advisors, the Banker to the Issue; and (b) Book Running Lead Managers, Syndicate Members, Escrow Collection Banker and Registrar to the Issue, to act in their respective capacities, have been obtained and shall be filed along with a copy of the Red Herring Prospectus with the Designated Stock Exchange and such consents have not been withdrawn up to the time of delivery of this Red Herring Prospectus for registration with the Designated Stock Exchange. M/s. Surendar K Jain & Co, M/s. Mookherjee Biswas & Pathak, M/s M.C. Bhandari & Co., M/s P.K.Chopra & Co, M/s. Ramanlal G Shah & Co, and M/s. B.K.Ramadhyani & Co, Chartered Accountants, our Auditors, have given their written consent to the inclusion of their report in the form and context in which it appears in this Red Herring Prospectus and such consent and report has not been withdrawn up to the time of delivery of this Red Herring Prospectus for filing with the Designated Stock Exchange. Minimum Subscription If we do not receive the minimum subscription of 90% of the Net Issue to the extent of the amount including devolvement of the members of the Syndicate, if any, within 60 days from the Bid Closing Date/Issue Closing Date, we shall forthwith refund the entire subscription amount received. If there is a delay beyond eight days after we become liable to pay the amount, we shall pay interest at the same rates as prescribed under Section 73 of the Companies Act. Method of Redressal of Investor Grievances See section titled “Other Regulatory Disclosures - Mechanism for Redressal of Investor Grievances” on page 209. Expert Opinion Except as stated elsewhere in this Red Herring Prospectus, we have not obtained any expert opinions. Changes in Auditors The Auditors of our Bank are being appointed by the Reserve Bank of India and his/their remuneration, rights and duties shall be regulated by the Bank Acquisition Act. There have been no changes of the auditors in the last three years except as detailed below: Year Name of the Statutory Auditors Fresh induction during Retired during this Reasons this Year year 2001-2002 M/s Mookherjee Biswas & Pathak M/s Mookherjee M/s M Bhaskara Completion of term M/s Surender K Jain & Co. Rao & Co. Biswas & Pathak Completion of term M/s SBA Associates M/s Surender K Jain & M/s V Soundaranjan M/s G P Agarwal & Co. Co. & Co. M/s Parekh & Co. M/s Kalani & Co. 2002-2003 M/s P K Chopra & Co. M/s P K Chopra & Co. M/s SBA Associates Completion of term M/s M C Bhandari & Co. M/s M C Bhandari & Co. M/s G P Agarwal & Completion of term M/s Kalani & Co. Co. M/s Mookherjee Biswas & Pathak M/s Parekh & Co. M/s Surender K Jain & Co. 243 2003-2004 M/s G P Kapadia & Co. M/s G P Kapadia & Co. M/s Parakh & Co. Completion of term M/s Kalani & Co. M/s Mookherjee Biswas & Pathak M/s M C Bhandari & Co. M/s P K Chopra & Co. M/s Surender K Jain & Co Basis of Allocation A. For Retail Individual Bidders Bids received from the Retail Individual Bidders at or above the Issue Price shall be grouped together to determine the total demand under this portion. The Allotment to all the successful Retail Individual Bidders will be made at the Issue Price. The Net Issue size less allocation to Non-Institutional Bidders and QIB Bidders shall be available for allocation to Retail Individual Bidders who have bid in the Issue at a price that is equal to or greater than the Issue Price. If the aggregate demand in this portion is less than or equal to 22,400,000 Equity Shares at or above the Issue Price, full Allotment shall be made to the Retail Individual Bidders to the extent of their demand. If the aggregate demand in this category is greater than 22,400,000 Equity Shares at or above the Issue Price, the allocation shall be made on a proportionate basis up to a minimum of [ ] Equity Shares and in multiples of [ ] Equity Shares thereafter. For the method of proportionate basis of allocation, refer below. B. For Non-Institutional Bidders Bids received from Non-Institutional Bidders at or above the Issue Price shall be grouped together to determine the total demand under this portion. The Allotment to all successful Non-Institutional Bidders will be made at the Issue Price. The Net Issue size less allocation to QIB Bidders and Retail Individual Bidders shall be available for allocation to Non- Institutional Bidders who have bid in the Issue at a price that is equal to or greater than the Issue Price. If the aggregate demand in this category is less than or equal to 9,600,000 Equity Shares at or above the Issue Price, full Allotment shall be made to Non-Institutional Bidders to the extent of their demand. In case the aggregate demand in this category is greater than 9,600,000 Equity Shares at or above the Issue Price, allocation shall be made on a proportionate basis up to a minimum of [ ] Equity Shares and in multiples of [ ] Equity Shares thereafter. For the method of proportionate basis of allocation refer below. C. For QIB Bidders Bids received from the QIB Bidders at or above the Issue Price shall be grouped together to determine the total demand under this portion. The allocation to all the QIB Bidders will be made at the Issue Price. The Net Issue size less allocation to Non-Institutional Bidders and Retail Individual Bidders shall be available for allocation to QIB Bidders who have bid in the Issue at a price that is equal to or greater than the Issue Price. The allocation would be decided by us in consultation with the BRLMs and would be at our sole discretion, based on various factors, such as quality of the Bidder, size, price and date of the Bid. The aggregate allocation to QIB Bidders shall not be more than 32,000,000 Equity Shares. D. For Employees Bids received from the Employees at or above the Issue Price shall be grouped together to determine the total demand in this portion. The Allotment to all the Employees who Bid successfully will be made at the Issue Price. If the aggregate demand in this portion is less than or equal to 8,000,000 Equity Shares at or above the Issue Price, full Allotment shall be made to the Employees to the extent of their demand. If the aggregate demand in this portion is greater than 8,000,000 Equity Shares at or above the Issue Price, the allocation shall be made on a proportionate basis subject to a minimum of [ ] Equity Shares. For the method of proportionate basis of allocation, please refer below. 244 E. For Existing Shareholders Bids received from the Existing Shareholders at or above the Issue Price shall be grouped together to determine the total demand in this portion. The Allotment to all the Existing Shareholders who Bid successfully will be made at the Issue Price. If the aggregate demand in this portion is less than or equal to 8,000,000 Equity Shares at or above the Issue Price, full Allotment shall be made to the Existing Shareholders to the extent of their demand. If the aggregate demand in this portion is greater than 8,000,000 Equity Shares at or above the Issue Price, the allocation shall be made on a proportionate basis subject to a minimum of [ ] Equity Shares. For the method of proportionate basis of allocation, please refer below. Method of proportionate basis of allocation in the Retail and Non-Institutional Portions Bidders will be categorized according to the number of Equity Shares applied for by them. (a) The total number of Equity Shares to be Allotted to each portion as a whole shall be arrived at on a proportionate basis, which is the total number of Equity Shares applied for in that portion (number of Bidders in the portion multiplied by the number of Equity Shares applied for) multiplied by the inverse of the over-subscription ratio. (b) Number of Equity Shares to be Allotted to the successful Bidders will be arrived at on a proportionate basis, which is total number of Equity Shares applied for by each Bidder in that portion multiplied by the inverse of the over-subscription ratio. In all Bids where the proportionate Allotment is less than [ ] Equity Shares per Bidder, the Allotment shall be made as follows: Each successful Bidder shall be Allotted a minimum of [ ] Equity Shares; and The successful Bidders out of the total Bidders for a portion shall be determined by draw of lots in a manner such that the total number of Equity Shares Allotted in that portion is equal to the number of Equity Shares calculated in accordance with (b) above; and. Each successful Bidder shall be Allotted a minimum of [ ] Equity Shares If the proportionate Allotment to a Bidder is a number that is more than [ ] but is not a multiple of one (which is the market lot), the decimal would be rounded off to the higher whole number if that decimal is 0.5 or higher. If that number is lower than 0.5, it would be rounded off to the lower whole number. Allotment to all Bidders in such categories would be arrived at after such rounding off. If the Equity Shares allocated on a proportionate basis to any portion are more than the Equity Shares allotted to the Bidders in that portion, the remaining Equity Shares available for Allotment shall be first adjusted against any other portion, where the Equity Shares are not sufficient for proportionate Allotment to the successful Bidders in that portion. The balance Equity Shares, if any, remaining after such adjustment will be added to the portion comprising Bidders applying for minimum number of Equity Shares. Expenses of the Issue The expenses of this Issue include, among others, underwriting and management fees, selling commission, printing and distribution expenses, legal fees, statutory advertisement expenses and listing fees. The estimated Issue expenses are as follows: Activity Expense(In Rs. millions) Lead management, underwriting and selling commission Rs. [ ] Advertisement & Marketing expenses Rs. 71.40 Printing, stationery including transportation of the same Rs. 39.50 Others(Registrar’s fees, Legal fee, listing fee, etc.) Rs. 112.2 Total estimated Issue expenses Rs. [ ] * Will be incorporated after finalisation of Issue Price 245 Fees Payable to the BRLMs, Brokerage and Selling Commission The total fees payable to the BRLMs including brokerage and selling commission for the Issue will be as per the memorandum of understanding executed between the Bank and the BRLMs dated January 13, 2005, a copy of which are available for inspection at our Head Office. Fees Payable to the Registrar to the Issue The fees payable to the Registrar to the Issue will be as per the Registrar’ memorandum of understanding dated January 28, 2005 copies of which is available for inspection at our Head Office. Adequate funds will be provided to the Registrar to the Issue to enable them to send refund orders or Allotment advice by registered post. Previous Rights and Public Issues For details of the previous public issues by our Bank, see the section titled “Other Regulatory Disclosures” on page 209. Commission and Brokerage paid on Previous Equity Issues by us Name of issue Month and Year Commission and Brokerage (in Rs.million) Public Issue of Equity Shares April, 2002 Rs. 13,823,389 Issues otherwise than for cash We have not issued any Equity Shares for consideration otherwise than for cash. Application in Issue Equity Shares being issued through this Red Herring Prospectus can be applied for in the dematerialized form only. Purchase of Property Except as stated in the section titled “Objects of the Issue” and elsewhere in this Red Herring Prospectus, there is no property which we have purchased or acquired or propose to purchase or acquire which is to be paid for wholly, or in part, from the net proceeds of the Issue or the purchase or acquisition of which has not been completed on the date of this Red Herring Prospectus, other than property in respect of which: the contracts for the purchase or acquisition were entered into in the ordinary course of the business, and the contracts were not entered into in contemplation of the Issue nor is the Issue contemplated in consequence of the contracts; or the amount of the purchase money is not material; or disclosure has been made earlier in this Red Herring Prospectus Except as stated in the section titled “Related Party Transactions” on page 149, we have not purchased any property in which any Directors, have any direct or indirect interest in any payment made thereof. Remuneration of Whole Time Directors 1. Mr. S.S.Kohli, Chairman and Managing Director: Mr. S.S.Kohli was appointed as Chairman and Managing Director of our Bank on April 20, 2000 by Central Government after consultation with the Reserve Bank of India under clause (a) of Sub-Section (3) of Section 9 of the Bank Acquisition Act for a period of five years. 246 The details of Mr. Kohli’s remuneration during the period from April 1, 2003 to March 31, 2004 is as below: (Rs.) Salary and allowances 498,900 Bank’s contribution to provident fund 31,200 Medical expenses 34,908 Club fees 4,380 Total 569,338 Other perquisites and benefits: In addition to the above, Mr. Kohli is entitled to medical benefits, leave travel assistance, entertainment allowance, city compensatory allowance, residential accommodation and conveyance. 2. Dr. K. C. Chakrabarty, Executive Director: Dr. K. C. Chakrabarty was appointed as Executive Director of our Bank on August 27, 2004 by the Government after consultation with the Reserve Bank of India under clause (a) of sub-section (3) of Section 9 of the Bank Acquisition Act for a period of five years or until further orders, whichever is earlier. The details of Dr. Chakrabarty’s remuneration with effect from August 27, 2004 to September 30, 2004 is as follow: (Rs.) Salary and Allowances 42,983 Bank’s Contribution to Provident Fund 3,841 Total 46,824 Other perquisites and benefits: In addition to the above, Dr. Chakrabarty is entitled to medical benefits, leave travel assistance, entertainment allowance, city compensatory allowance, residential accommodation and conveyance. Expenditure Incurred By The Directors (Excluding The Whole Time Directors) The expenditure incurred on traveling, lodging and fees paid to the Directors for the period from April 1, 2003 to March 31, 2004 is Rs. 3.49 million. Capitalisation of Reserves or Profits We have not capitalised our reserves or profits at any time. Revaluation of Assets We have not revalued our assets in the past five years. Payment or benefit to officers of our Bank No amount or benefit has been paid or given within the two preceding years or is intended to be paid or given to any of our officers except the normal remuneration for services rendered as Directors, officers or employees. There is no service contract for the directors providing for benefits on termination on service. 247 MAIN PROVISIONS OF CONSTITUTIONAL DOCUMENTS We were constituted as a “corresponding new bank” in 1970 under the provisions of the Bank Acquisition Act. We are not required to have memorandum and articles of association. Since we were constituted under the Bank Acquisition Act we have profiled the salient terms of the Act. Further since the Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970 and the Bank Regulations deal with the management of corporate affairs in our Company, which are matters typically finding a place in the constitutional documents of a company incorporated under the Companies Act, the same have been profiled in this chapter. The Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970 was made by S.O. 3793 dated November 16, 1970 by the central government in consultation with the Reserve Bank of India in exercise of the powers conferred by Section 9 of the Bank Acquisition Act. The Bank Regulations were formulated under Section 19 of the Bank Acquisition Act by our board of directors in consultation with the Reserve Bank of India, and with the previous sanction of the central government. The Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 amended Section 34A, 36AD and Section 51 of the Banking Regulation Act, 1949 and made these sections applicable to corresponding new banks constituted under the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970. For details of the applicability of the Banking Regulation Act to correspondent new bank like our Bank see the section titled “Regulations and Policies” on page 151. For complete text of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970, Banking Regulation Act, 1949, Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970 and the Punjab National Bank (Shares and Meetings) Regulations, 2000 as on the date of filing of this Red Herring Prospectus please see the link to our Constitutional Documents on www.pnbindia.com, which is the website of the Bank. Shareholders and investors in the Bank may note that the rights available to shareholders of a corresponding new bank are more restricted than the rights available to the shareholders of a company incorporated under the Companies Act, 1956. For further details on the restrictions and their potential impact on shareholders of our Bank please see the section titled “Risk Factors – External Risk Factors” on page xxii. The salient features of the same are as below. BANKING COMPANIES (ACQUISITION AND TRANSFER OF UNDERTAKINGS) ACT, 1970 3. Establishment of corresponding new banks and business thereof. (1) On the commencement of this Act, there shall be constituted such corresponding new banks as are specified in the First Schedule. (2) The paid-up capital or every corresponding new bank constituted under sub-section (1) shall, until any provision is made in this behalf in any scheme made under Section 9, be equal to paid-up capital of the existing bank in relation to which it is the corresponding new bank. (2A) Subject to the provisions of this Act, the authorised capital of every corresponding new bank shall be one thousand five hundred crores of rupees divided into one hundred fifty crores fully paid-up shares of ten rupees each. Provided that the Central Government may, after consultation with the Reserve Bank and by notification in the Official Gazette, increase or reduce the authorised capital as it thinks fit, so however that after such increase or reduction, the authorised capital shall not exceed three thousand crores or be less than one thousand five hundred crores, of rupees. (2B) Notwithstanding anything contained in sub-section (2), the paid-up capital of every corresponding new bank constituted under sub-section (1) may from time to time be increased by :— (a) such amounts as the Board of Directors of the corresponding new bank may, after consultation with the Reserve Bank and with the previous sanction of the Central Government, transfer from the reserve fund established by such bank to such paid-up capital; (b) such amounts as the Central Government may, after consultation with the Reserve Bank, contribute to such paid- up capital; 248 (c) such amounts as the Board of Directors of the corresponding new bank may, after consultation with the Reserve Bank and with the previous sanction of the Central Government, raise by public issue of shares in such manner as may be prescribed, so however that the Central Government shall, at all times, hold not less than fifty-one per cent of the paid-up capital of each corresponding new bank. (2BB) Notwithstanding anything contained in sub-section (2), the paid-up capital of a corresponding new bank constituted under sub-section (1) may, from time to time and before any paid-up capital is raised by public issue under clause (c) of sub-section (2B), be reduced by- (a) the Central Government, after consultation with the Reserve Bank, by canceling any paid-up capital which is lost, or is unrepresented by available assets; (b) the Board of Directors, after consultation with the Reserve Bank and with the previous sanction of the Central Government, by paying off any paid-up capital which is in excess of the wants of the corresponding new bank. Provided that in a case where such capital is lost, or is unrepresented by available assets because of amalgamation of another corresponding new bank or a corresponding new bank as defined in clause (d) of Section 2 of the Banking Companies (Acquisition arid Transfer of Undertakings) Act. 1970 (5 of 1970) with the corresponding new bank, such reduction may be done, either prospectively or retrospectively, but not from a date earlier than the date of such amalgamation. (2BBA)(a) A corresponding new bank may from time to time and after any paid-up capital has been raised by public issue under clause (c) of sub-section (2B), by resolution passed at an annual general meeting of the shareholders entitled to vote, voting in person, or, where proxies are allowed, by proxy, and the votes cast in favour of the resolution are not less than three times the number of the votes, if any, cast against the resolution by the shareholders so entitled and voting, reduce its paid-up capital in any way. (b) without prejudice to the generality of the foregoing power the paid-up capital may be reduced by:— (i) extinguishing or reducing the liability on any of its shares in respect of share capital not paid-up; (ii) either with or without extinguishing or reducing liability on any of its paid-up shares, cancelling any paid-up capital which is lost, or is unrepresented by available assets; or (iii) either with or without extinguishing or reducing liability on any of its paid-up shares, paying off any paid share capital which is in excess of the wants of the corresponding new bank. (2BBB) Notwithstanding anything contained in sub-section (2BB) or sub-section (2BBA), the paid-up capital of a corresponding new bank shall not be reduced at any time so as to render it below twenty-five per cent of the paid-up capital of that bank as on the date of commencement of the Banking Companies (Acquisition and Transfer of Undertakings) Amendment Act, 1995.” (2C) The entire paid-up capital of a corresponding new bank, except the paid-up capital raked by public issue under clause (c) of sub-section (2B), shall stand vested in and allotted to the Central Government. (2D) The shares of every corresponding new bank not held by the Central Government shall be freely transferable: Provided that no individual or company resident outside India or any company incorporated under any law not in force in India or any branch of such company, whether resident outside India or not, shall at any time hold or acquire by transfer or otherwise shares of the corresponding new bank so that such investment in aggregate exceed the percentage, not being more than twenty per cent of the paid-up capital as may be specified by the Central Government by notification in the Official Gazette. Explanation— For the purposes of this clause “company” means any body corporate and includes a firm or other association of individuals. (2E) No shareholder of the corresponding new bank, other than the Central Government, shall be entitled to exercise voting rights in respect of any shares held by him in excess of one per cent of the total voting rights of all the shareholders of the corresponding new bank. 249 (2F) Every corresponding new bank shall keep at its head office a register in one or more books, of the shareholders (in this Act referred to is the register) and shall enter therein the following particulars (i) the names, addresses and occupations, if any, of the shareholders and a statement of the shares held by each shareholder, distinguishing each share by its denoting number; (ii) the date on which each Person is so entered as a shareholders; (iii) the date on which any Person ceases to be a shareholder; and (iv) such other particulars as may be prescribed. (2G) Notwithstanding anything contained in sub-section (2F), it shall be lawful for every corresponding new bank to keep the register in computer floppies or diskettes subject to such safeguards as may be prescribed. (3) Notwithstanding anything contained in the Indian Evidence Act, 1872 (1 of 1872) a copy of, or extract from, the register, certified to be a true copy under the hand of an officer of the corresponding new bank authorised in this behalf by it, shall, in all legal proceedings, be admissible in evidence. (4) Every corresponding new bank shall be a body corporate with perpetual succession and a common seal with power, subject to the provisions of this Act, to acquire, hold arid dispose of property, and to contract, and may sue and be sued in its name. (5) Every corresponding new bank shall carry on and transact the business banking as defined in clause (b) of Section 5 of the Banking Regulation Act, 1949 (10 of 1949), and may engage in (one or more of the other forms of business specified in sub-section (1) of Section 6 of that Act. (6) Every corresponding new bank shall establish a reserve fund to which shall be transferred to share premiums arid the balance, if any, standing to the credit of the reserve fund of the existing bank in relation to which it is the corresponding new bank, and such further sums, if any, as may be transferred in accordance with the provisions of Section 17 of the Banking Regulation Act, 1949 (10 of 1949). (7) (i) The corresponding new bank shall, if so required by the Reserve Bank, act as agent of the Reserve Bank at all places in India where it has a branch, for- (a) paying, receiving, collecting and remitting money, bullion any securities on behalf of any Government in India; and (b) undertaking and transacting any other business which the Reserve Bank may from time to time entrust to it. (ii) The terms and conditions on which any such agency business shall be carried on by the corresponding new bank on behalf of the Reserve Bank shall be such as may be agreed upon. (iii) If no agreement can be reached on any matter referred to in clause (ii), or if a dispute arises between the corresponding new bank and the Reserve Bank as to the interpretation of any agreement - between them, the matter shall be referred to the Central Government and the decision of the Central Government thereon shall be final. (iv) The corresponding new bank may transact any business or perform any functions entrusted to it under clause (a), by itself or through any agent approved by the Reserve Bank. 7. Head office and management (1) The head office of each corresponding new bank shall be at such place as the Central Government may, by notification in the Official Gazette, specify in this behalf, and, until any such place is so specified, shall be at such place at which the head office of the existing bank, in relation to which it is the corresponding new bank, is on the commencement of this Act, located. (2) The general superintendence, direction and management of the affairs and business of a corresponding new bank shall vest lit a Board of Directors which shall be entitled to exercise all such powers and do all such acts and things as the corresponding new bank is authorised to exercise and do. (3) (a) As soon as may be after the appointed day, the Central Government shall, in consultation with the Reserve Bank, constitute the first Board of Directors of a corresponding new bank, consisting of not more than seven 250 Persons, to be appointed by the Central Government, and every Director so appointed shall hold office until the Board of Directors of such corresponding new bank is constituted in accordance with the scheme made under Section 9: Provided that the Central Government may, if it is of opinion that it is necessary in the interests of the corresponding new bank so to do, remove a Person from the Membership of the first Board of Directors and appoint any other Person in this place. (b) Every Member of the first Board of Directors (not being an officer of the Central Government or of the Reserve Bank) shall receive such remuneration as is equal to the remuneration which a Member of the Board of Directors of the existing bank was entitled to receive immediately before the commencement of this Act. (4) Until the first Board of Directors is appointed by the Central Government under sub-section (3), the general superintendence, direction and management of the affairs and business of a corresponding new hank shall vest in a Custodian, who shall be the Chief Executive Officer of that bank and may exercise all powers and do all acts things as may be exercised or done by that bank. (5) The Chairman of an existing bank holding office as such immediately before the commencement of this Act, shall be the Custodian of the corresponding new bank and shall receive the same emolument as he was receiving immediately before such commencement: Provided that the Central Government may, if the Chairman of an existing bank declines to become, or to continue to function as, a Custodian of the corresponding new bank, or, if it is of opinion that it is necessary in the interests of the corresponding new bank so to do, appoint any other Person as the Custodian of a corresponding new bank and the Custodian so appointed shall receive such emoluments as the Central Government may specify in this behalf. (6) The Custodian shall hold office during the pleasure of the Central Government. 8. Corresponding new banks to be guided by the Directions of the Central Government Every corresponding new bank shall, in the discharge of its functions, be guided by such directions in regard to matters of policy involving public interest as the Central Government may, after consultation with the Governor of the Reserve Bank, give. 9. Power of Central Government to make scheme (1) The Central Government may, after consultation with the Reserve Bank, make a scheme for carrying out the provisions of this Act. (2) In particular, and without prejudice to the generality of the foregoing power, the said scheme may provide for all or any of the following matters, namely:— (a) the capital structure of the corresponding new bank; (b) the constitution of the Board of Directors, by whatever name called, of the corresponding new bank and all such matters in connection therewith or incidental thereto as the Central Government may consider to be necessary or expedient; (c) the reconstitution of any corresponding new bank into two or more corporations, the amalgamation of arty corresponding new bank with any other corresponding new bank or with another banking institution, the transfer of the whole or any part of the undertaking of a corresponding new bank to any other corresponding new bank or banking institution or the transfer of the whole or any part of the undertaking of any other banking institution to a corresponding new bank; (d) such incidental, consequential and supplemental matters as may be necessary to carry out the provisions of this Act. (3) Every Board of Director of a corresponding new bank constituted under any scheme made under sub-section (1), shall include— (a) not more than two whole-time Directors to be appointed by the Central Government after consultation with the Reserve Bank; 251 (b) one Director who is an official of the Central Government to be nominated by the Central Government: Provided that no such Director shall be a Director of any other corresponding new bank. Explanation—For the purposes of this clause, the expression corresponding new bank” shall include a corresponding new bank within the meaning of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1980 (40 of 1980); (c) one Director who is an Officer of the Reserve Bank to be nominated by the Central Government on the recommendation of the Reserve Bank. Explanation.—For the purpose of this clause, “an Officer of the Reserve Bank” includes an officer of the Reserve Bank who is deputed by that Bank under Section 54AA of the Reserve Bank of India Act, 1934 (2 of 1934) to any institution referred to therein; (d) not more than two Directors to be nominated by the Central Government from amongst the Securities and Exchange Board of India established under Section 3 of the Securities and Exchange hoard of India Act, 1992 (15 of 1992), the National Bank for Agriculture and Rural Development established under Section 3 of the National Bank for Agriculture and Rural Development Act, 1981 (61 of 1981), public financial institutions as specified in sub-section (1), or notified from time to time under sub-section (2) of Section 4A of the Companies Act, 1956 (1 of 1956) and other institutions established or constituted by or under arty Central Act or incorporated under the Companies Act, 1956 and having not less than fifty one per cent of the paid- up share capital held or controlled by the Central Government; (e) one Director, from among such of the employees of the corresponding new bank who are workmen under clause (s) of Section 2 of the Industrial Disputes Act. 1947 (14 of 1947) to be nominated by the Central Government in such manner as may be specified in a scheme made under this section; (f) one Director, from among the Employees of the corresponding new bank who are not workmen under clause (s) of Section 2 of the Industrial Disputes Act, 1947, (14 of 1947) to be nominated by the Central Government after consultation with the Reserve Bank; (g) one Director who has been a Chartered Accountant for not less than fifteen years to be nominated by the Central Government after consultation with the Reserve Bank; (h) subject to the provisions of clause (i), not more than six Directors to be nominated by the Central Government; (i) where the capital issued under clause (c) of sub-section (2B) of Section (3) is— (I) not more than twenty per cent of the total paid-up capital, not more than two Directors. (II) more than twenty per cent but not more than forty per cent of the total paid-up capital, not more than tour Directors, (III) more than forty per cent of the total paid-up capital, not more than six Directors, to be elected by the shareholders, other than the Central Government from amongst themselves: Provided that on the assumption of charge after election of any such Directors under this clause, equal number of Directors nominated under clause & (h) shall retire in such manner as may be specified in the scheme. (3A) The Directors to be nominated under clause (h) or to be elected under clause (1) ofsub-section (3) shall— (A) have special knowledge or practical experience in respect of one or more of the following matters namely :- (i) agricultural and rural economy, (ii) banking, (iii) economics, (iv) co-operation, (v) finance, (vi) law, (vii) small-scale industry, 252 (viii) any other matter the special knowledge of, arid practical experience in, which would, in the opinion of the Reserve Bank, he useful to the corresponding new bank; (B) represent the interests of depositors ; or (C) represent the interests of farmers, workers and artisans. (3B) Where the Reserve Bank is of the opinion that any Director of a corresponding new bank elected under clause (1) of sub-section (3) does not fulfill the requirements of sub-section (3A), it may, after giving to such Director and the bank a reasonable opportunity of being heard, by order, remove such Director and on such removal, the Board of Directors shall co-opt any other person fulfilling the requirements of sub-section (3A) as a Director in place of the person so removed till a Director is duly elected by the shareholders of the corresponding new bank in the next annual general meeting and the person so, co-opted shall be deemed to have been duly elected by the shareholders of the corresponding new bank as a Director. (4) The Central Government may, alter consultation with the Reserve Bank, make a scheme to amend or vary any scheme made under sub-section (1). (5) On and from the date of coming into operation of a scheme made under this section with respect to any of the matters referred to in clause (c) of sub-section (2) or any matters incidental, consequential and supplemental thereto,— (a) the scheme shall be binding on the corresponding new bank or corporations or banking institutions, and also on the Members, if any, the depositors, and other creditors and Employees of each of them and on any other persons having any right or liability in relation to any of them including the trustees or other persons, managing or in any other manner connected with any provident fund or other fund maintained by any of them; (b) the properties and assets of the corresponding new bank, or as the case may be, of the banking institution shall, by virtue of and to the extent provided in the scheme, stand transferred to, and vested in, and the liabilities of the corresponding new bank, or, as the case may be, of the banking institution shall, by virtue of, and to the extent provided in the scheme, stand transferred to, and become the liabilities of, the corporation or corporations brought into existence by reconstitution of the banking institution or the corresponding new bank, as the case may be. Explanation I— In this section, “banking institution” means ‘a banking company and includes the State Bank of India or a subsidiary bank. Explanation II— For the purposes of this section, the expression “corresponding new bank” shall include a corresponding new bank within the meaning of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1980 (40 of 1980). 10. Closure of accounts and disposal of profits (1) Every corresponding new bank shall cause its books to be closed and balanced on the 31st day of December or such other date in each year as the Central Government may, by notification In the Official Gazette, specify and shall appoint, with the previous approval of the Reserve Bank, Auditors for the audit of the audit of its accounts: Provided that with a view to facilitating the transition from one period of accounting to another period of accounting tinder this sub-section, the Central Government may, by order published in the Official Gazette, make such provisions as it considers necessary or expedient for the closing and balancing of. or for other matters relating to, the books in respect of the concerned years. (2) Every Auditor of a corresponding new bank shall be a person who is qualified to -act as an Auditor of a company under Section 226 of the Companies Act, 1956 (1 of 1956) and shall receive such remuneration as the Reserve Bank may fix in consultation with the Central Government. (3) Every Auditor shall be supplied with a copy of the annual balance sheet and profit and loss account and a list of all books kept by the corresponding new bank, and it shall be the duty of the Auditor to examine the balance-sheet 253 and profit and loss account with the accounts and vouchers relating thereto, and in the performance of his duties, the Auditor:— (a) shall have, at all reasonable times, access to the books, accounts and other documents of the corresponding new bank, (b) may, at the expense of the corresponding new bank, employee accountants or other persons to-assist him in investigating such accounts, and (c) may, in relation to such accounts, examine the Custodian or any Officer or Employee of the corresponding new bank. (4) Every Auditor of a corresponding new bank shall make a report to the Central Government upon the annual balance sheet and accounts and in every such report shall state: (a) whether, in his opinion, the balance-sheet is a full and fair balance-sheet containing all the necessary particulars arid is properly drawn up so as to exhibit a true and fair view of the affairs of the corresponding new bank, and in case he had called for any explanation or information, whether it has been given and whether it is satisfactory; (b) whether or not the transactions of the corresponding new bank, which have come to his notice, have been within the powers of that bank; (c) whether or not the returns received from the offices and branches of the corresponding new bank have been found adequate for the purpose of his audit; (d) whether the profit and loss account shows a true balance of profit or loss for the period covered by such account ; and (e) any other matter which he considers should be brought to the notice of the Central Government. Explanation I: For the purposes of this Act (a) the balance-sheet shall not be treated as not disclosing a true and fair view of the affairs of the corresponding new bank, and (b) the profit and lose account shall not be treated as not showing a true balance of profit or loss for the period covered by such account, merely by reason of the fact that the balance-sheet or, as the case may be, the profit and loss account, does not disclose any matters which arc by the provisions of the Banking Regulation Act 1949 (10 of 1949), read with the relevant provisions of this Act or any other Act, not required to be disclosed. Explanation II—For the purposes of this Act the accounts of the corresponding new bank shall not be deemed as having not been properly drawn up on the ground merely that they do not disclose certain matters if: (i) those matters are such as the corresponding new bank is, by virtue of any provision contained in the Banking Regulation Act, 1949 (1 of 1949), read with the relevant provisions of this Act, or any other Act, not required to disclose ; and (ii) the provisions referred to in clause (i) are specified in the balance sheet and profit and loss account of the corresponding new bank or in the Auditor’s report. (5) The report of the Auditor shall be verified, signed and transmitted to the Central Government. (6) The Auditor shall also forward a copy of the audit report to the corresponding new bank and to the Reserve Bank. (7) After making provision for bad and doubtful debts, depreciation in assets, contributions to staff and superannuation funds and all other matters for which provision is necessary under any law, or which are usually provided for by banking companies, a corresponding new bank may out of its net profits deal an a dividend and retain the surplus if any. (7A) Every corresponding new bank shall furnish to the Central Government to the Reserve Bank the annual balance sheet, the profit and loss account, and the Auditor’s report and a report by its Board of Directors or the working and activities of the bank during the period covered by the accounts 254 (8) The Central Government shall cause every Auditors report and report on the working and activities of each corresponding new bank to be laid as soon as may be after they are received before each House of Parliament. (9) Without prejudice to the foregoing provisions, the Central Government may, at any time, appoint such number of Auditors as it thinks fit to examine and report on the accounts of a corresponding new bank and the Auditors so appointed shall have all the rights, privileges and authority it relation to the audit of the accounts of the corresponding new bank which an Auditor appointed by the corresponding new bank has under this section. 10A. Annual general meeting (1) A general meeting (in this Act referred to as an annual general meeting) of every corresponding new bank which has issued capital under clause (c) of sub-section (2B) of Section 3 shall be held at the place of the head office of the bank in each year at such time as shall from time to time be specified by the Board of Directors: Provided that such annual general meeting shall be held before the expiry of six weeks from the date on which the balance sheet, together with the profit and loss account and Auditor’s report is under sub-section (7A) of Section 10, forwarded to the Central Government or to the Reserve Bank whichever date is earlier. (2) The shareholders present at an annual general meeting shall be entitled to discuss the balance-sheet and the profit and loss account of the corresponding new bank made up to the previous 31st day of March, the report of the Board of Directors on the working and activities of the corresponding new bank for the period covered by the accounts and the Auditor’s report on the balance-sheet and accounts. 11. Corresponding new bank deemed to be an Indian company For the purposes of the Income-tax Act, 1961 (43 of 1961), every corresponding new bank shall be deemed to be an Indian company and a company in which the public are substantially interested. 15. Certain defects not to invalidate acts of proceedings (l) All acts done by the Custodian, acting in good faith, shall, notwithstanding any defect in his appointment or in the procedure, be valid. (2) No act or proceeding of any Board of Directors or a local Board or Committee of a corresponding new bank shall be invalid merely on the ground of the existence of any vacancy in, or defect in the constitution of, such Board or be Committee, as the case may be. (3) All acts done by a person acting in good faith as a Director or Member of a local Board or Committee of a corresponding new bank shall be valid notwithstanding that it may afterwards be discovered that his appointment was not invalid by reason of any defect or disqualification or had terminated by virtue of any provision contained In any law for the time being in force: Provided that nothing in this section shall be deemed to give validity to any act by a Director or Member of a local Board or Committee of a corresponding new bank after his appointment has been shown to the corresponding new bank to be invalid or to have terminated. 16. Indemnity (1) Every custodian of a corresponding new bank and every Officer of the Central Government or of the Reserve Bank and every Officer or other Employee of a corresponding new bank, shall be indemnified by such bank against all losses and expenses incurred by him in or in relation to the discharge of his duties except such as have been caused by his own willful act or default. (2) A Director or Member of a local Board or Committee of a corresponding new bank shall not be responsible for any loss or expense caused to such bank by the insufficiency or deficiency of the value of, or title to, any property or security acquired or taken on behalf of the corresponding new bank, or by the insolvency or wrongful act of any customer or debtor, or by anything done in or in relation to the execution of the duties of his office, unless such loss, expense, insufficiency or deficiency was due to any willful act or default on the part of such Director or Member. 255 16A. Arrangement with corresponding new bank on appointment of Directors to prevail (1) Where any arrangement entered into by a corresponding new bank with a company provides for the appointment by the corresponding new bank of one or more Directors of such Company, such appointment of Directors made in pursuance thereof shall be valid and effective notwithstanding anything to the contrary contained in the Companies Act, 1956 (1 of 1956) or in any other law for the time being in force or in the memorandum, articles of association or any other instrument relating to the Company, and any provision regarding share qualification, age limit, number of Directorship, removal from office of Directors and such like conditions contained in any such law or instrument aforesaid, shall not apply to any Director appointed by the corresponding new bank in pursuance of the arrangement as aforesaid. (2) Any Director appointed as aforesaid shall— (a) hold office during the pleasure of the corresponding new bank and may be removed or substituted by any person by order in writing of the corresponding new bank; (b) and incur any obligation or liability by reason only of his being a Director or for anything done or omitted to be done in good faith in the discharge of his duties as a Director or anything in relation thereto; (c) not be liable to retirement by rotation and shall not be taken into account for computing the number of Directors liable to such retirement. (d) Power to make regulations (3) The Board of Directors of a corresponding new bank may, after consultation with the Reserve Bank and with the previous sanction of the Central Government, by notification in the Official Gazette make regulations, not inconsistent with the provisions of this Act or any scheme made thereunder, to provide for all matters for which provision is of a expedient for the purpose of giving effect to the provisions of this Act. (4) Until any regulation is made under sub-section (1), the articles of association of the existing bank and every regulation, rule, bye-law or order made by the existing bank shall, if in force at the commencement of this Act, be deemed to be the regulations made under sub-section (1) and shall have effect accordingly and any reference therein to any authority of the existing bank shall be deemed to be a reference to the corresponding authority of the corresponding new bank and until any such corresponding authority is constituted under this Act, shall be deemed to refer to the Custodian. (5) Every regulation shall, as soon as may be after it is made under this Act by the Board of Directors of a corresponding new bank, be forwarded to the Central Government and that Government shall cause a copy of the same to be laid before each House of Parliament, while it is in session, for a total period of thirty days which may be comprised in one session or in two or more successive sessions, and if, before the expiry of the session immediately following the session or the successive sessions aforesaid, both houses agree in making any modification in the regulation or both Houses agree that the regulation should not be made, the regulation shall thereafter have effect only in such modified form or be of no effect, as the case may be; so, however, that any such modification or annulment shall be without prejudice to the validity of anything previously done under that regulation. NATIONALISED BANKS (MANAGEMENT AND MISCELLANEOUS PROVISIONS) SCHEME, 1970 3. Constitution of the Board (2) (i) The director referred to in Clause (e) of sub-section (3) of Section 9 of the Act, shall be nominated by the Central Government from out of a panel of three such employees furnished to it by the representative union, within a date to be specified by the Central Government, which date shall not be more than six weeks from the date of communication made by the Central Government, requiring the representative union to furnish the panel of names: Provided that where the Central Government is of the opinion that owing to the delay which is likely to occur in the verification and certification of any union or federation as a representative union it is necessary in the interest of the Nationalised Bank so to do, it may nominate any employee of the Nationalised Bank, who is a workman, to be a director of that bank. 256 (ii) (a) Where there is no representative union, to represent the workman of a Nationalised Bank, or (b) where such representative union being in existence omits or fails to furnish any panel of names within the specified date, or (c) where all the persons specified in the panel furnished by the representative union are disqualified whether under item (iii) of this sub-clause or under Clause 10, the Central Government may, at its discretion appoint such workman of the Nationalised Bank, as it may think fit, to be a director of such bank. (iii) A workman of a Nationalised Bank shall be disqualified for being nominated as a director unless— (a) he is, and has been serving for a continuous period of not less than five years in the Nationalised Bank; and (b) he is of such age that there is no likelihood of his attaining the age of superannuation during his terms of office as director. 4. Manner of retirement of nominee directors The director referred to in Clause (h) of sub-section (3) of Section 9 of the Act shall retire by rotation, when the elected directors assume charge, in such manner that the directors who have been longest in office since the last nomination, shall retire first and as between persons, who became directors on the same day, those who are to retire, shall, in default of or subject to any agreement among themselves, be decided by the Central Government. 5. Chairman (1) The Central Government shall, after consultation with the Reserve Bank, appoint one of the Directors to be the Chairman of the Board. (2) The Chairman shall preside over the meetings of the Board. 6. Managing Director The Central Government shall, after consultation with the Reserve Bank, appoint one of the directors referred to in Clause (a) of sub-section (3) of Section 9 of the Act to be the Managing Director, who shall be the Chief Executive Officer of the Nationalised Bank and shall exercise the powers and discharge such duties as may be delegated to him by the Board. 7. Same person may hold office as Chairman and Managing Director The Central Government may, after consultation with the Reserve Bank appoint the same person to hold, at the same time, both the office of the Chairman and the Managing Director. 8. Term of office and remuneration of a whole-time Director including Managing Director (1) A whole-time Director, including the Managing Director shall devote his whole-time to the affairs of the affairs of the Nationalised Bank and shall hold office for such terms not exceeding five years as the Central Government may, after consultation with the Reserve Bank, specify and shall be eligible for re-appointment. (1-A) Notwithstanding anything contained in sub-clause (1), the Central Government shall have the right to terminate the term of office of a whole-time Director, including the Managing Director, at any time before the expiry of the term specified under that sub-clause by giving to him a notice of not less than three months, in writing or three months salary and allowances in lieu of notice; and the whole-time Director, including the Managing Director, shall also have the right to relinquish his office at any time before the expiry of the term specified under that sub- clause by giving to the Central Government notice of not less than three months in writing. (1-B) Any reference to a whole-time Director, including the Managing Director, in sub-clause (1-A) shall be construed as including a reference to the person holding office as such at the commencement of the Nationalised Banks (Management and Miscellaneous Provision (Second Amendment) Scheme, 1976. (2) A whole-time Director, including the Managing director shall receive from the Nationalised Bank such salary, allowance, fees and perquisites and be governed by such terms and conditions as the Central Government may determine, after consultation with the Reserve Bank. (3) If a whole-time Director including the Managing Director is by infirmity or otherwise rendered incapable of carrying out his duties or is absent on leave or otherwise in circumstances not involving the vacation of his office, 257 the Central Government may, after consultation with the Reserve Bank, appoint another person to act in his place during his absence. (4) The Central Government may, if it is satisfied that it is expedient in the interests of the nationalized bank so to do, remove a whole-time Director including the Managing Director from office: Provided that no such removal shall be made except after— (a) consultation with the Board, and (b) giving a reasonable opportunity to the whole-time Director including the Managing director, of showing cause against the proposed action. 9. Term of office of other directors (1) A director other than a director referred to in Clause (a) and Clause (i) of sub-section (3) of Section 9 of the Act shall hold office during the pleasure of the Central Government. (2) a) Subject to the provisions of sub-clause (1), a director referred to in Clause (e), Clause (f), Clause (g) and Clause (h) of sub-section (3) of Section 9 of the Act shall hold office for such term not exceeding three years as the Central Government may specify at the time of his nomination and thereafter until his successor has been nominated and shall be eligible for re-nomination (b) a director referred to in clause (g) and (h) of sub-section (3) of section 9 of the Act shall hold office for such term not exceeding three years as the Central Government may specify at the time of his nomination and shall be eligible for re-nomination. Provided that no such director shall hold office continuously for a period of six years. Provided that no such director shall hold office continuously for a period exceeding six years. (3) Without prejudice to the provisions of sub-clauses (1) and (2), a director referred to in Clause (b) of sub-section (3) of Section 9 of the Act shall retire in the manner specified in Clause 4. (4) An elected director shall hold office for three years and shall be eligible for re-election: Provided that no such director shall hold office continuously for a period exceeding six years. 10. Disqualification of Directors A person shall be disqualified for being appointed as, and for being, a director— (a) if he has at any time been adjudicated an insolvent or has suspended payment or has compounded with his creditors; or (b) if he has been found to be of unsound mind and stands so declared by a competent Court; or (c) if he has been convicted by a Criminal Court of an offence which involves moral turpitude. 11. Vacation of office of Directors, etc. (1) If a director becomes subject to any of the disqualifications specified in Clause 10 or is absent without leave of the Board for more than three consecutive meetings thereof he shall be deemed to have vacated his office as such and thereupon his office shall become vacant. (2) The Chairman or whole-time director including the Managing Director or a director referred to in Clause (b) or Clause (c) or Clause (d) of sub-section (3) of Section 9 of the Act may resign his office by giving notice thereof in writing to the Central Government and on such resignation being accepted by that Government shall be deemed to have vacated his office; any other director may resign his office by giving notice thereof in writing to the Central Government and such resignation shall take effect on the receipt of the communication of the resignation by the Central Government. (3) Without prejudice to the provisions of the foregoing sub-clause, the office of a director referred to in Clause (e) or Clause (f) of sub-section (3) of Section 9 of the Act shall become vacant as soon as the director ceases to be a workman or an employee, other than a workman of the nationalized bank of which he is a director. (4) Where any vacancy occurs in the office of a director, other than an elected director, it shall be filled in accordance with sub-section (3) of Section 9 of the Act. 258 11-A. Removal from office of an elected director The shareholders other than the Central Government, may, by a resolution passed by majority of the votes of such shareholders holding in the aggregate not less than one half of the share capital held by all such shareholders, remove any director elected under Clause (i) of sub-section (3) of Section 9 and elect in his stead another person to fill the vacancy. 11-B. Filling of vacancy in the office of an elected director (1) Where any vacancy occurs before the expiry of the term of office of an elected director, the vacancy shall be filled in by election: Provided that where the duration of vacancy is likely to be less than six months, the vacancy may be filled in by the remaining directors. (2) A person elected or cooperated, as the case may be, under sub-clause (1) shall hold office for the unexpired portion of the term of his predecessor. 12. Meetings of the Board (1) Meetings of the Board shall ordinarily be held at least six times in a year and at least once in each quarter. (2) A meeting of the Board shall be held at the head office of the nationalised bank or such other place as the Board may decide. (3) Ordinarily, not less than fifteen days’ notice shall be given of any meeting of the Board and such notice shall be sent to every director at the address specified by him in this behalf. (4) No business, other than that for which the meeting was convened shall be transacted at a meeting of the Board except with the consent of the Chairman of the meeting and a majority of the directors present, unless one week’s notice of such business has been given in writing to the Chairman. (5) The quorum of a meeting of the Board shall be one-third of the number of directors holding office as such directors of the Board on the day of the meeting, subject to a minimum of three directors, two of whom shall be directors referred to in Clause (b) or Clause (c) or Clause (d) or Clause (h) of sub-section (3) of Section 9 of the Act. (6) If, for any reason, the Chairman is unable to attend a meeting of the Board, the Managing Director shall preside over that meeting and in the absence of the Managing Director or in the event of the Chairman and the Managing Director being the same person, any other director elected by the directors present at the meeting from among themselves shall preside at the meeting. (7) All questions at the meeting shall be decided by a majority of the votes of the directors present and voting and in the case of equality of votes, the person presiding shall have a second or a casting vote. (8) A director who is directly or indirectly concerned or interested in any contract, loan, arrangement or proposal entered into or proposed to be entered into by or on behalf of the nationalized bank shall, as soon as possible after the relevant circumstances have come to his knowledge, disclose the nature of his interest to the Board and shall not be present at the meeting of the Board when any such contract, loan, arrangement or proposal is discussed unless his presence is required by the other directors for the purpose of eliciting information and no director so required to be present shall vote on any such contract, loan, arrangement or proposal: Provided that nothing contained in this sub-clause shall apply to such director by reason only of his being— (i) a shareholder (other than a director) holding not more than two per cent of the paid-up capital in any public company as defined in the Companies Act, 1956 (1 of 1956), or any corporation established by or under any law for the time being in force in India or any co-operative society, with which or to which the Nationalised Bank has entered into or made or proposed to enter into or make, a contract, loan, arrangement or proposal, or (ii) an officer or other employee of the nationalized bank, if he is a director referred to in Clause (e) or Clause (f) of sub-section (3) of Section 9 of the Act. (9) A copy of the proceedings of each meeting of the Board shall be circulated as soon as possible after the meeting for other information of the directors and shall be signed by the Chairman of that or the next succeeding meeting. 259 (10) No act or proceeding of the Board shall be invalid on the ground merely of the existence of any vacancy in or any defect in the constitution of the Board. 13. Management Committee (1) There shall be a Management Committee of the Board. (2) The Management Committee shall consist of— (A) The Chairman; (B) The Managing Director; (C) The Executive Directors; (D) The Directors referred to in Clauses (b), (c) and (g) of sub-section (3) of Section 9 of the Act; (E) One Director nominated by the Board from amongst, the directors referred to in Clause (d) of sub-section (3) of Section 9 of the Act; and (F) One Director nominated by the Board from amongst the Directors referred to in Clauses (e), (f), (h) and (i) of sub-section (3) of Section 9 of the Act. Provided that the Directors nominated by the Board shall hold office for not more than six months at a time. (3) The Management Committee shall exercise such powers of the Board including the powers with regard to credit proposals, as may be delegated to it by the Board with the approval of the Central Government and such approval shall be given by the Central Government after consultation with the Reserve Bank of India. (4) The meetings of the Management Committee may be called by the Chairman of the Management Committee as often as he feels necessary. (5) Four members shall be the quorum for a meeting of the Management Committee. (6) The minutes of a meeting of the Management Committee shall be laid before the Board as soon as possible after the meeting. (7) Save as otherwise provided in sub-clauses (4), (5) and (6) the meetings and proceedings of the Management Committee shall be governed by the provisions contained in this Scheme for regulating the meetings and proceedings of the Board so far as the same are applicable thereto. (8) Where the Chairman of the Management Committee is of opinion that in view of urgency in any matter, it should be dealt with expeditiously, he may circulate a resolution to that effect to the members of the Management Committee, and such resolution shall be deemed to be the resolution passed by the Management Committee when it is approved by a majority of the Members but shall have effect from the date it is signed by the last signatory to the resolution: Provided that any resolution passed as aforesaid shall be placed before the next meeting of the Management Committee: Provided further that if any dissenting member requires in writing that any resolution so passed shall be placed before a meeting of the Management Committee, the resolution shall not be deemed to be valid and effectual as aforesaid unless the same is passed at such meeting. Explanation— For the purpose of sub-clause (2), “Executive Director” means the whole-time Director, not being the Managing Director, appointed under sub-clause (a) of Clause 3 and designated as such. 18. Resolution without meeting of the Board valid A resolution in writing signed by the majority of the members of the Board shall be valid and effectual and shall be deemed to be the resolution passed by the Board on the date it was signed by the last signatory to the resolution: Provided that any resolution passed as aforesaid shall be placed before the next meeting of the Board: Provided further that if any dissenting member requires in writing that any resolution so passed shall be placed before a meeting of the Board, the resolution shall not be deemed valid and effectual as aforesaid unless the same is passed at such meeting. 260 20. Increase of paid-up capital The paid-up capital of a Nationalised Bank may be increased from time to time as in sub-clause (a) or sub-clause (b) or sub-clause (c) below or in combination with any of them: (a) the Board of Directors of a Nationalised Bank may, after consultation with the Reserve Bank and with the previous sanction of the Central Government transfer to its capital a specified amount from the reserve fund establishment by such bank under sub-section (6) of Section 3 of the Act; (b) the Central Government may, in consultation with the Reserve Bank, make contribution of any specified amount to the paid-up capital of a Nationalised Bank; (c) the Board may, after consultation with the Reserve Bank and with the previous sanction of the Central Government, raise the paid-up capital by public issue of shares in such manner as may be prescribed; so however, that the Central Government shall at all times hold not less than fifty-one per cent of the paid-up capital of each Nationalised Bank. PUNJAB NATIONAL BANK (SHARES & MEETINGS) REGULATIONS, 2000 Definition 2(a) “Act” means the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 (5 of 1970); 3. Nature of Shares The shares of the Bank shall be movable property, transferable in the manner provided in the Bank Regulations 4. Kinds of Share Capital (i) Preference Share Capital means that part of share capital of the Bank which fulfils both the following conditions: (A) as respects dividends, it carries a preferential right to be paid a fixed amount or an amount calculated at fixed rate, which may be either free of or subject to income tax; and (B) as respect capital, it carries or will carry, on winding up to repayment of capital, a preferential right to be repaid the amount of the capital paid-up or deemed to have been paid up, whether or not there is preferential right to the payment of either or both of the following amounts, namely: a) any money remaining unpaid, in respect of the amounts specified in clause (A) upto the date of winding up or repayment of capital, and b) any fixed premium or premium on any fixed scale, specified by the Board with the previous consent of the Central Government. (ii) “Equity Share Capital” means all share capital, which is not preference share capital. (iii) The expressions “Preference Share” and “Equity Share” shall be construed accordingly. 5. Particulars to be entered in the register (i) A share register shall be kept, maintained and updated in, accordance with Sub-Section 2 (F) of Section 3 of the Act. (ii) In addition to the particulars specified in sub-section 2 (F) of Section 3 of the Act, such other particulars as the Board may specify shall be entered in the register. (iii) In the case of joint holders of any share, their names and other particulars required by sub-regulation (i) shall be grouped under the name of the first of such joint holders. (iv) Subject to the proviso of sub-section 2 (D) of sec. 3 of the Act, a shareholder resident outside India may furnish to the Bank an address in India and any such address shall be entered in the register and be deemed to be his registered address for the purposes of the Act and these regulations. 261 7. Parties who may not be registered as shareholders Except as otherwise provided by the Bank, all persons who are not competent to contract shall not be entitled to be registered as a shareholder and the decision of the board of directors of the Bank (“Board”) in this regard shall be conclusive and final. (i) In case of partnership firms, shares may be registered in the names of the individual partners and no firm as such, shall be entitled to be registered as a shareholder. 10. Exercise of rights of joint holders If any share stands in the names of two or more persons, the person first named in the register shall as regards voting, receipt of dividends, service of notices and all or any other matters connected with the Bank except the transfer of shares, be deemed to be the sole holder thereof. 11. Inspection of Register The register of shareholders, except when closed under the Bank Regulations, shall be open to inspection of any shareholder, free of charge, at the place where it is maintained during business hours subject to such reasonable restrictions as the Board may impose, but so that not less than two hours in each working day shall be allowed for inspection. 13. Share Certificates (i) Each share certificate shall bear share certificate number, a distinctive number, the number of shares in respect of which it is issued and the name of the shareholder to whom it is issued and it shall be in such form as may be specified by the Board. (ii) Every share certificate shall be issued under the common seal of the Bank and shall be singed by two directors and some other officer not below the rank of scale II or the company secretary appointed by the Board for the purpose. (iii) No share certificate shall be valid unless and until it is so signed. 15. Issue of new or duplicate share certificates (i) If any share certificate is worn out or defaced, the Board or the committee designated by it on production of such certificate may order the same to be cancelled and have a new certificate issued in lieu thereof. (ii) If any share certificate is alleged to be lost or destroyed, the Board or the committee designated by it, on such indemnity with or without surety as the Board or the committee thinks fit, and on publication in two newspapers and on payment to the Bank of its costs, charges and expenses, a duplicate certificate in lieu thereof may be given to the person entitled to such lost or destroyed certificate. 16. Consolidation and sub-division of shares On a written application made by the shareholder(s), the Board or the committee designated by it may consolidate or sub-divide the shares submitted to it for consolidation/ sub-division as the case may be and issue new certificate(s) in lieu thereof on payment to the Bank of its costs, charges and expenses of and incidental to the matter. 17. Transfer of shares (i) Every transfer of the shares of the Bank shall be by an instrument of transfer in the prescribed form and shall be duly stamped, dated and executed by or on behalf of the transferor and the transferee alongwith the relative share certificate. (ii) The instrument of transfer alongwith the share certificate shall be submitted to the Bank at its head officer and the transferor shall be deemed to remain the holder of such shares until the name of the transferee is entered in the share register in respect thereof. (iii) Upon receipt by the Bank of an instrument of transfer alongwith a share certificates with a request to register the transfer, the Board or the committee designated by the Board shall forward the said instrument of transfer alongwith share certificate to the registrar or share transfer agent for the purposes of verification that the technical requirements are complied with in their entirety. The registrar or share transfer agent shall return the instrument 262 of transfer alongwith the share certificate, if any, to the transferee for resubmission unless the instrument of transfer is presented to the Bank, duly stamped and properly executed for registration and is accompanied by the certificate of the shares to which it relates and such other evidence as the Board may require to show the title of the transferor to make such transfer. 18. Power to suspend transfers The Board or the committee designated by the Board shall not register any transfer during any period in which the register is closed. 19. Board’s right to refuse registration of transfer of shares (i) The Board or committee may refuse transfer of any shares in the name of the transferee on any one or more of the following grounds, and on no other grounds: a. the transfer of shares is in contravention of the provisions of the Bank Acquisition Act or regulations made thereunder or any other law or that any other requirement under the law relating to registration of such transfer has not been complied with; b. the transfer of shares, in the opinion of the Board, is prejudicial to the interests of the Bank or to public interest; c. the transfer of shares is prohibited by an order of court, tribunal or any other authority under any law for the time being in force; d. an individual or a company resident outside India or any company incorporated under any law not in force in India or any branch of such company, whether resident outside India or not, will on the transfer being allowed, hold or acquire as a result thereof, shares of the Bank and such investment in the aggregate will exceed the percentage being more than 20% of the paid up capital or as may be prescribed by the central government by notification in the official gazette. (ii) The Board or the committee shall, after the instrument of transfer of shares of the Bank is lodged with it for the purpose of registration of such transfer, form its opinion as to whether such registration ought or ought not to be refused on any of the grounds referred to above: e. if it has formed the opinion that such registration ought not to be so refused, effect such registration; and if it has formed the opinion that such registration ought to be refused on any of the grounds mentioned above intimate the same to the transferor and the transferee by notice in writing giving reasons for such refusal within 60 days from the receipt of the transfer form or within such period as may be laid down in the listing agreement with the concerned stock exchange. 20. Transmission of shares in the event of death, insolvency etc.: (i) The executors or administrators of a deceased share holder in respect of a share, or the holder of letter of probate or letters of administration with or without the will annexed or a succession certificate issued under Part X of the Indian Succession Act, 1925, or the holder of any legal representation or a person in whose favour a valid, instrument of transfer was executed by the deceased sole holder during the latter’s lifetime shall be the only person who may be recognised by the Bank as having any title to such share. (ii) In the case of shares registered in the name of two or more shareholders, the survivor or survivors and on the death of the last survivor, his executors or administrators or any person who is the holder of letters of probate or letters of administration with or without will annexed or a succession certificate or any other legal representation in respect of such survivor’s interest in the share or a person in whose favour a valid instrument of transfer of share was executed by such person and such last survivor during the latter’s lifetime, shall be the only person who may be recognised by the Bank as having any title to such share. (iii) The Bank shall not be bound to recognise such executors or administrators unless they shall have obtained probate or letters of administration or succession certificate, as the case may be, from a court of competent jurisdiction. Provided, however, that in a case where the Board in its discretion thinks fit, it shall be lawful for the Board to dispense with the production of letters of probate or letters of administration or succession certificate or such 263 other legal representation, upon such terms as to indemnity or otherwise as it may think fit. (iv) Any such person becoming entitled to a share in consequence of death of a shareholder and any person becoming entitled to a share in consequence of the insolvency, bankruptcy or liquidation of a shareholder shall upon production of such evidence, as the Board may require, have the right a) to be registered as a shareholder in respect of such share. b) to make such transfer of such share as the person from whom he derives title could have made. 21. Shareholder ceasing to be qualified for registration It shall be the duty of any person registered as a shareholder, whether solely or jointly with another or others forthwith upon ceasing to be qualified to be so registered in respect of any share to give intimation thereof to the Board of Directors in this regard. Explanation - For the purposes of this regulation, a shareholder may cease to be qualified for registration, - (a) If he is a guardian of minor, on the minor attaining the majority; (b) If he is holding shares as a Karta, on his ceasing to be a Karta. 22. Calls on shares The Board may, from time to time, make such calls as it thinks fit upon the shareholders in respect of all moneys remaining unpaid on the shares held by them, which are by the conditions of allotment not made payable at fixed times, and each shareholder shall pay the amount of every call so made on him to the person and at the time and place appointed by the Board. A call may be payable by installments. 23. Calls to date from resolution A call shall be deemed to have been made at the time when the resolution of the Board authorising such call was passed and may be made payable by the shareholders on the register on such date or at the discretion of the Board on such subsequent date as may be fixed by the Board. 24. Notice of call A notice of not less than thirty days of every call shall be given specifying the time of payment provided that before the time for payment of such call the Board may by notice in writing to the shareholders revoke the same. 25. Extension of time for payment of call The Board may, from time to time and at its discretion, extend the time fixed for the payment of any call to all or any of the shareholders having regard to distance of their residence or some other sufficient cause, but no shareholder shall be entitled to such extension as a matter of right. 26. Liabilities of joint holders The joint holders of a share shall be jointly and severally liable to pay all calls in respect thereof. 29. Non-payment of calls by shareholder No shareholder shall be entitled to receive any dividend or to exercise any right of a shareholder until he shall have paid all calls for the time being due and payable on every share held by him, whether singly or jointly with any person, together with interest and expenses, as may be levied or charged. 30. Notice on non-payment of call or instalment If any shareholder fails to pay the whole or any part of any call or instalment or any money due in respect of any shares either by way of principal or interest on or before the day appointed for the payment of the same, the Bank may at any time thereafter during such time as the call or instalment or any part thereof or other moneys remain unpaid or a judgement or decree in respect thereof remains unsatisfied in whole or in part, serve a notice on such shareholder or on the person (if any) entitled to the share by transmission, requiring him to pay such call or instalment or such part thereof or other moneys as remain unpaid together with any interest that may have accrued and all expenses (legal or otherwise) that may have been paid or incurred by the Bank by reason of such non-payment. 264 31. Notice of Forfeiture The notice of forfeiture shall name a day not being less than fourteen days from the date of the notice and the place or places on and at which such call or instalment or such part or other monies and such interest and expenses as e aforesaid are to be paid. The notice shall also state that in the event of non-payment on or before the time and at the place appointed, the share in respect of which the call was made or instalment is payable will be liable to be forfeited. 32. Shares to be forfeited on default If the requirement of any such notice as aforesaid are not complied with, any of the shares in respect of which such notice has been given may at any time thereafter for non-payment of all calls or instalments, interest and expenses or the money due in respect thereof, be forfeited by a resolution of the Board to that effect at its next meeting to be held after the expiry of the notice of forfeiture under regulation 31. Such forfeiture shall include all dividends declared in respect of the forfeited shares and not actually paid before the forfeiture. 33. Entry of forfeiture in the register When any share has been forfeited under Regulation 32, an entry of the forfeiture with the date thereof shall be made in the register. 34. Forfeited shares to be property of the Bank and may be sold Any share so forfeited shall be deemed to be the property of the Bank and may be sold, re-allotted or otherwise disposed of to any person upon such terms and in such manner as the Board may decide. 35. Powers to annul forfeiture The Board may, at any time, before any share so forfeited under Regulation 32 shall have been sold, re-allotted or otherwise disposed of, annul the forfeiture thereof upon such conditions as it may think fit. 36. Shareholder liable to pay money owing at the time of forfeiture and interest Any shareholder whose shares have been forfeited shall, notwithstanding the forfeiture, be liable to pay shall and forthwith pay to the Bank all calls, instalments, interest, expenses and other moneys owing upon or in respect of such shares at the time of forfeiture with interest thereon from the time of forfeiture until payment at such rate as may be specified by the Board and the Board may enforce the payment of the whole or a portion thereof. 37. Partial payment not to preclude forfeiture Neither a judgement nor a decree in favour of the Bank for calls or other monies due in respect of any shares nor any payment or satisfaction thereunder nor the receipt by the Bank of a portion of any money which shall be due from any shareholder from time to time in respect of any shares either by way of principal or interest nor any indulgence granted by the Bank in respect of payment of any money shall preclude the forfeiture of such shares under these regulations. 38. Forfeiture of share extinguishes all claims against Bank The forfeiture of a share shall involve extinction, at the time of the forfeiture, of all interest in and all claims and demands against the Bank, in respect of the share and all other rights incidental to the share, except only such of those rights as by these presents expressly waived. 39. Original shares null and void on sale, re-issue, re-allotment or disposal on being forfeited Upon any sale, re-issue, re-allotment or other disposal under the provision of the preceding regulations certificate(s) originally issued in respect of the relative shares shall (unless the same shall on demand by the Bank have been previously surrendered to it by the defaulting member) stand cancelled and become null and void and of no effect, the Board shall be entitled to issue a new certificate or certificates in respect of the said shares to the person or persons entitled thereto. 40. Application of forfeiture provisions The Provisions of these regulations as to the forfeiture shall apply in the case of non-payment of any sum which by terms of issue of a share become payable at a fixed time, whether on account of nominal value of the shares or by way of premium as if the same had been payable by virtue of a call duly made. 265 41. Lien on shares (i) The Bank shall have a first and paramount lien - a) on every share (not being a fully-paid share), for all moneys (whether presently payable or not) called, or payable at a fixed time, in respect of that share. b) on all shares (not being fully-paid shares), standing registered in the name of a single person, for all moneys presently payable by him or his estate in the Bank. c) upon all the shares registered in the name of each person (whether solely or jointly with others) and upon the proceeds of sale thereof for his debts, liabilities, and engagements, solely or jointly with any other person to or with the Bank, whether the period for the payment, fulfillment, or discharge thereof shall have actually arrived or not and no equitable interest in any share shall be recognized by the Bank over its lien. Provided that the Board of Directors may at any time declare any share to be wholly or in part exempt from provisions of this clause. (ii) The Bank’s lien, if any, on a share shall extend to all dividends payable thereon. 42. Enforcing Lien by Sale of Shares - (i) The Bank may sell, in such manner as the Board thinks fit, any shares on which the company has a lien: a) if a sum in respect of which the lien exists is presently payable, and b) after the expiration of fourteen days after a notice in writing stating and demanding payment of such part of the amount in respect of which the lien exists as is presently payable, has been given to the registered holder for the time being of the share or the person entitled thereto by reason of his death or insolvency. (ii) To give effect to any such sale, the Board may authorise some officer to transfer the shares sold to the purchaser thereof. 43. Application of proceeds of sale of shares The net proceeds of any sale of shares under Regulation 42 after deduction of costs of such sale, shall be applied in or towards the satisfaction of the debt or liability in respect whereof the lien exists so far as the same is presently payable and the residue, if any, be paid to the shareholders or the person, if any, entitled by transmission to the shares so sold. 44. Certificate of forfeiture A certificate in writing under the hands of any director, or Company Secretary or any other officer of the Bank not below the rank of Scale II duly authorised in this behalf, that the call in respect of a share was made that the forfeiture of the share was made by a resolution of the Board to that effect, shall be conclusive evidence of the fact stated therein as against all persons entitled to such shares. 45. Title of purchaser and allottee of forfeited share The Bank may receive the consideration, if any, given for the share on any sale, re-allotment or other disposition thereof and the person to whom such share is sold, re-allotted or disposed of may be registered as the holder of the share and shall not be bound to see to the application of the consideration, if any, nor shall his title to the share be affected by any irregularity or invalidity in the proceedings in reference to the forfeiture, sale, re-allotment or other disposal of the share and the remedy of any person aggrieved by the sale shall be in damages only and against the Bank exclusively. 56. Notice convening an Annual General Meeting (i) A notice convening an annual general meeting of the shareholders signed by the Chairman and Managing Director or Executive Director or any officer not below the rank of Scale VII or Company Secretary shall be published at least twenty one clear days before the meeting in not less than two daily newspapers having wide circulation in India. (ii) Every such notice shall state the time, date and place of such meeting and also the business that shall be transacted at that meeting. (iii) The time and date of such meeting shall be as specified by the Board. The Meeting shall be held at the place of head office of the Bank. 266 58. Quorum of general meeting (i) No business shall be transacted at any meeting of the shareholders unless a quorum of at least five shareholders entitled to vote at such meeting in person are present at the commencement of such business. (ii) If within half an hour after the time appointed for the holding of a meeting, a quorum is not present in the case of a meeting called by a requisition of shareholders other than the Central Government, the meeting shall stand dissolved. (iii) In any other case if within half an hour after the time appointed for the holding of a meeting, a quorum is not present, the meeting shall stand adjourned to the same day in the next week, at the same time and place or to such other day and such other time and place as the Chairman may determine. If at the adjourned meeting a quorum is not present within half an hour from the time appointed for holding the meeting, the shareholders who are present in person or by proxy or by duly authorised representative at such adjourned meeting shall be quorum and may transact the business for which the meeting was called: Provided that no annual general meeting shall be adjourned to a date later than the date within which such annual general meeting shall be held in terms of Section 10A(l) of the Act and if adjournment of the meeting to the same day in the following week would have this effect, the annual general meeting shall not be adjourned but business of the meeting shall be commenced within one hour from the time appointed for the meeting if the quorum is present or immediately after the expiry of one hour from that time and those shareholders who are present in person or by proxy or by duly authorised representative at such time shall form the quorum. 61. Voting at general meetings (i) At any general meeting, a resolution put to the vote of the meeting shall, unless a poll is demanded be decided on a show of hands. (ii) Save as otherwise provided in the Act every matter submitted to a general meeting shall be decided by a majority of votes. (iii) Unless a poll is demanded under sub-regulation (i), a declaration by the Chairman of the meeting that a resolution on show of hands has or has not been carried either unanimously or by a particular majority and an entry to that effect in the books containing the minutes of the proceedings shall be conclusive evidence of the fact, without proof of the number or proportion of the votes cast in favour of, or against, such resolution. (iv) Before or on the declaration of the result of the voting on any resolution on a show of hands, a poll may be ordered to be taken by the chairman of the meeting of his own motion and shall be ordered to be taken by him on a demand made in that behalf by any shareholder or shareholders present in person or by proxy and holding shares in the Bank which confer a power to vote on the resolution not being less than one fifth of the total voting power in respect of the resolution. (v) The demand for a poll may be withdrawn at any time by the person or persons who made the demand. (vi) A poll demanded on a question of adjournment or election of chairman of the meeting shall be taken forthwith. (vii) A poll demanded on any other question shall be taken at such time not being later than forty eight hours from the time when the demand was made, as the Chairman of the meeting may direct. (viii) The decision of the chairman of the meeting as to the qualification of any person to vote, and also in the case of poll, as to the number of votes any person is competent to exercise shall be final. 61B. Manner of taking poll and result thereof: (i) The Chairman of the meeting shall have power to regulate the manner in which a poll shall be taken. (ii) The result of the poll shall be deemed to be the decision of the meeting on the resolution on which the poll was taken. 63. Directors to be elected at general meeting - (i) A director under clause (i) of sub-section (3) of Section 9 of the Act shall be elected by the shareholders on the register, other than the Central Government, from amongst themselves in the general meeting of the Bank. 267 (ii) Where an election of a director is to be held at any general meeting, the notice thereof shall be included in the notice convening the meeting. Every such notice shall specify the number of directors to be elected and the particulars of vacancies in respect of which the election is to be held. 65. Nomination of candidates for election (i) No nomination of a candidate for election as a director shall be valid unless, a. he is a shareholder holding not less than 100 (one hundred) shares in the Bank; b. he is on the last date for receipt of nomination, not disqualified to be a director under the Act or under the Scheme; c. he has paid all calls in respect of the shares of the Bank held by him, whether alone or jointly with others, on or before the last date fixed for the payment of the call; d. the nomination is in writing signed by at least one hundred shareholders entitled to elect directors under the Act or by their duly constituted attorney, provided that a nomination by a shareholder who is a company may be made by a resolution of the directors of the said company and where it is so made, a copy of the resolution certified to be a true copy by the Chairman of the meeting at which it was passed shall be despatched to the Head Office and such copy shall be deemed to be a. nomination on behalf of such company. e. the nomination accompanies or contains a declaration signed by the candidate before a Judge, Magistrate, Registrar or Sub-Registrar of Assurances or other Gazetted Officer or an Officer of the Reserve Bank of India or any nationalised Bank, that he accepts the nomination and is willing to stand for election and that he is not disqualified either under the Act or the Scheme or these regulations from being a director. (ii) No nomination shall be valid unless it is received with all the connected documents complete in all respects and received, at the Head Office on a working day not less than fourteen days before the date fixed for the meeting. 66. Scrutiny of nominations (i) Nominations shall be scrutinised on the first working day following the date fixed for receipt of the nominations and in case any nomination is not found to be valid, the same shall be rejected after recording the reason therefor. If there is only one valid nomination for any particular vacancy to be filled by election, the candidate so nominated shall be deemed to be elected forthwith and his name and address shall be published as so elected. In such an event, there shall not be any election at the meeting convened for the purpose and if the meeting had been called solely for the purpose of the aforesaid election, it shall stand cancelled. (ii) In the event of an election being held, if valid nominations are more than the number of directors to be elected, the candidate polling the majority of votes shall be deemed to have been elected. (iii) A director elected to fill an existing vacancy shall be deemed to have assumed office from the date following that on which he is, or is deemed to be elected. 67. Election Disputes (i) If any doubt or dispute shall arise as to the qualification or disqualification of a person deemed, or declared to be elected, or as to the validity of the election of a director, any person interested, being a candidate or shareholder entitled to vote at such election, may, within seven days of the date of the declaration of the result of such election, give intimation in writing thereof to the Chairman and Managing Director of the Bank and shall in the said intimation give full particulars of the grounds upon which he doubts or disputes the validity of the election. (ii) On receipt of intimation under sub-regulation (1), the Chairman and Managing Director or in his absence, the Executive Director of the Bank shall forthwith refer such doubt or dispute for the decision of a committee consisting of the Chairman and Managing Director or in his absence, the Executive Director and any two of the directors nominated under clauses (b) & (c) of sub-section (3) of Section 9 of the Act. (iii) The committee referred to in sub-regulation (ii) shall make such enquiry as it deems necessary and if it finds that the election was a valid election, it shall confirm the declared result of the election or, if it finds that the election was not a valid election, it shall, within 30 days of the commencement of the enquiry, make such order and give such directions including the holding of a fresh election as shall in the circumstances appear just to the committee. 268 (iv) An order and direction of such committee in pursuance of this regulation shall be conclusive. 68. Determination of voting rights (i) Subject to the provisions contained in Section 3 (2E) of the Act, each shareholder who has been registered as a shareholder on the date of closure of the register prior to the date of a general meeting shall, at such meeting, have one vote on show of hands and in case of a poll shall have one vote for each share held by him. (ii) Subject to the provisions contained in Section 3 (2E) of the Act, every shareholder entitled to vote as aforesaid who, not being a company, is present in person or by proxy or who being a company is present by a duly authorised representative, or by proxy shall have one vote on a show of hands and in case of a poll shall have one vote for each share held by him as stated hereinabove in sub-regulation (i) Explanation - For this Chapter, “Company” means any body corporate. (iii) Shareholders of the Bank entitled to attend and vote at a general meeting shall be entitled to appoint another person (whether a shareholder or not) as his proxy to attend and vote instead of himself but a proxy so appointed shall not have any right to speak at the meeting. 69. Voting by duly authorised representative - (i) A shareholder, being the Central Government or a company, may by a resolution, as the case may be, authorise any of its officials or any other person to act as its representative at any general meeting of the shareholders and the person so authorised (referred to as a ‘duly authorised representative” in these regulations) shall be entitled to exercise the same powers on behalf of the Central Government or company which he represents as if he were an individual shareholder of the Bank. The authorisation so given may be in favour of two persons in the alternative and in such a case any one of such persons may act as the duly authorised representative of the Central Government/ company. (ii) No person shall attend or vote at any meeting of the shareholders of the Bank as the duly authorized representative of a company unless a copy of the resolution appointing him as a duly authorized representative certified to be a true copy by the Chairman of the meeting at which it was passed shall have been deposited at the Head Office not less than four days before the date fixed for the meeting. 269 FINANCIAL STATEMENTS 1. Auditors Report dated January 29, 2005. 2. Unconsolidated Restated Financial Statements of Punjab National Bank as of and for the fiscal years 2000, 2001, 2002, 2003 and 2004 and the six month period ended September 30, 2004 and September 30, 2003 and notes thereto. Annexure A I: Summary Statement of Profit and Loss as Restated II: Summary Statement of Assets and Liabilities as Restated III: Statement of Cash Flow as Restated IV: Significant Accounting Policies as at 31st March, 2004 V: Notes to Adjustments Carried Out VI: Adjustments not carried out in the statement of profit & loss and assets & liabilities VII: Material Notes on Accounts VIII: Related Party Transactions IX: Segment Reporting Annexure B Statement of Dividends declared by the Bank 3. Annexure C: Financial Statements of PNB Gilts Limited and notes thereto 4. Annexure D: Financial Statements of PNB Housing Finance Limited and notes thereto 5. Annexure E: Financial Statements of PNB Assets Management Company Limited and notes thereto 6. Annexure F: Financial Statements of PNB Capital Services Limited and notes thereto 7. Annexure G: Financial Statements of PNB Securities Limited and notes thereto 8. Annexure H: Consolidated Financial Statements of Punjab National Bank and notesthereto for fiscal years 2002, 2003 and 2004 9. Annexure I: Summary of Accounting Ratios 10. Annexure J: Capitalization Statement 11. Annexure K: Statement of Tax Shelters 12. Annexure L: Statement of Borrowings F - 1 29th January 2005 The Board of Directors Punjab National Bank HO: Bhikhaiji Cama Place New Delhi-110066. Dear Sirs, We have been engaged to examine and report on the financial information of Punjab National Bank (the Bank), which have been prepared in accordance with the Securities and Exchange Board of India (Disclosure and investor Protection) Guidelines, 2000 issued by the Securities and Exchange Board of India (SEBI) on January 19, 2000 in pursuance of section 11 of the SEBI Act, 1992, (the SEBI Guidelines). The preparation and presentation of this financial information is the responsibility of Bank’s management. These financial information are proposed to be included in the offer document of the Bank in connection with its proposed follow on public offer of equity shares. 1. For our examination, we have placed reliance on the following: - a) The financial statements of the Bank for the financial years ended on March 31, 2000, 2001, 2002, 2003 and 2004 which were audited and reported upon by the respective auditors, names of whom and the period of their audit are furnished below: Year Name of Auditors 1999-00 M/s Raj K Aggarwal & Associates, S.Mann & Co, V.Soundararajan & Co, SBA Associates, G.P Agrawal & Co, Parakh & Co 2000-01 M/s SBA Associates, GP Agrawal & Co., Parakh & Co., V. Soundararajan & Co, Kalani & Co., M Bhaskara Rao & Co. 2001-02 M/s SBA Associates, GP Agrawal & Co., Parakh & Co., Kalani & Co., Surendar K Jain & Co., Mookherjee Biswas & Pathak 2002-03 M/s Parakh & Co., Kalani & Co., Surendar K Jain & Co, Mookherjee Biswas & Pathak, M C Bhandari & Co, P K Chopra & Co 2003-04 M/s Kalani & Co, Surendar K Jain & Co, Mookherjee Biswas & Pathak, M C Bhandari & Co, P K Chopra & Co, G P Kapadia & Co b) The financial statements of the Bank for the half-year ended 30th September 2004, was subject to a limited review by us vide our report dated 2nd December 2004. The aforesaid financial statements incorporated the relevant returns of 20 branches reviewed by us and unreviewed returns in respect of 3,978 branches and 58 other offices. In the conduct of our review, we had taken note of the review reports in respect of the non-performing advances received from the concurrent auditors of 774 branches. These review reports covered (i)69.34 % of the advances portfolio of the Bank excluding advances portfolio of asset recovery and outstanding food credit and (ii) 91.85 % of non- performing advances. c) The financial statements of the Bank were last reviewed upto 30th September 2004. Since no financial statements have been reviewed subsequent to 30th September 2004 till date, prior to three months before the issue of the prospectus, the said financial statements of the Bank for the half-year ended 30th September 2004 have been incorporated. 2 The audit of the financial statements for the periods referred to in paragraph 1(a) of this report comprised of audit tests and procedures deemed necessary by the respective auditors for the purpose of expressing an opinion on such financial statements taken as a whole in accordance with generally accepted auditing practices. 3. The said review of the financial statements for the period referred to in paragraph1 (b) above consisted principally of applying analytical procedures to financial data and making enquiries of persons responsible for financial and accounting matters. It is substantially less in scope than an audit conducted in accordance with generally accepted auditing standards, the objective of which is an expression of an opinion on the financial statements as a whole. Accordingly, neither was an audit performed nor an opinion expressed. F - 2 4. We have performed such tests and procedures, which, in our opinion, were necessary for the purpose of our examination. These procedures, mainly involved comparison of the attached financial information with the Bank’s audited financial statements for the years 1999-2000 to 2003-2004 and unaudited financial statements for the half-years ended 30th September 2003 and 2004 as prepared by the bank. 5. We report that the profits of the Bank as restated for the above periods are as set out in annexure A. These profits have been arrived at after charging all operating and management expenses, including depreciation and after making such adjustments and regroupings as in our opinion are appropriate and are subject to the accounting policies and notes thereon furnished. 6 We report that the assets and liabilities of the Bank as restated for the above periods are also as set out in annexure A after making such adjustments and regroupings as in our opinion are appropriate and are subject to accounting policies and notes thereon furnished. 7 Annexure A has been subdivided as under: - a) Summary statement of Profit and Loss as restated. b) Summary statement of Assets and Liabilities as restated. c) Statement of Cash Flows as restated for the years 2001-02, 2002-03 and 2003-04 and the half years ended 30th September 2003 and 2004. The Bank did not prepare Cash Flow Statements for the years 1999-2000 and 2000-01 since the Accounting Standard 3 issued by the Institute of Chartered Accounts of India was not mandatory for those years. d) Significant accounting policies as at 31st March 2004. e) Notes on adjustments carried out f) Adjustments not carried out in the Statements of Profits and Losses and Assets and Liabilities. g) Material notes on accounts. h) Related Party transactions. i) Segment Reporting for the years 2001-02, 2002-03, 2003-04 and the half-year ended 30th September 2004. The Bank did not prepare segment reports for the years 1999-2000 and 2000-01 since the relevant accounting standard was not mandatory for those years. 8. We report that the dividends (subject to deduction of tax at source, wherever applicable) declared by the Bank for the five financial years ended March 31, 2004 are as set out in annexure B. 9. In accordance with the SEBI Guidelines, please find attached in annexures C, D, & E summary financial statements of subsidiaries of the Bank - PNB Gilts Ltd, PNB Housing Finance Ltd and PNB Asset Management Co. Ltd for the five years ended March 31, 2004 and the half-year ended 30th September 2004, wherever available. We understand that on 30th April 2004, all schemes of Punjab National Bank Mutual Fund were transferred to the Principal Financial Group. As per business transfer agreement, PNB Asset Management Company Limited has received a retirement fees of Rs 32 million. We further understand that there is a proposal to merge PNB Asset Management Company Limited with the bank. 10. The summary financial statements of PNB Capital Services Ltd., an erstwhile subsidiary of the Bank for the four years ended 31st March 2003 as prepared by the bank are enclosed in annexure F. This company was amalgamated with the Bank vide orders of the honorable high court of Delhi dated 25th August, 2003, with 1st of April 2003 as the appointed day. 11. The summary financial statements of PNB Securities Limited, an erstwhile subsidiary of the Bank for the financial years ended 31st March 2000 and 31st March 2001 and the period ended 16th August 2001 as prepared by the bank are enclosed in annexure G. This company went into liquidation on 16th of August 2001. F - 3 12. The holdings of the Bank in each of the above subsidiaries are set out below: Name of the subsidiary Percentage of holding as on 31st March 2000 2001 2002 2003 2004 PNB Gilts Limited * 74.79 74.79 74.79 74.79 74.07 PNB Capital Services Limited 100 100 100 100 N.A. PNB Housing Finance Limited 100 100 100 100 100 PNB Asset Management Co. Limited 100 100 100 100 100 PNB Securities Limited 100 100 N.A. N.A. N.A. * including holdings of subsidiaries of the Bank. 13. Annexures C, D and E are as per reports of the respective auditors detailed under: Annexure number Name of subsidiary Name of auditor Report dated Annexure C PNB Gilts Limited Raj K. Aggrawal & Associates, 10th January 2005 Chartered Accountants Annexure D PNB Housing Finance Limited Grover, Lalla & Mehta, 13th October 2004 Chartered Accountants Annexure E PNB Asset Management Gupta Goel & Khanna 15th October 2004 Company Limited Chartered Accountants We have not carried out any audit or review of the same. Annexures F to G are as per the audited financial statements of two subsidiaries referred to therein and furnished to us by the bank. These have not been audited, reviewed or subject to adjustments by us. The profits, losses, assets and liabilities as per annexures C to G relate to the entire individual subsidiaries. 14. We set out in annexure H a summary of the consolidated assets, liabilities, income and expenditure of the Bank (as a group) for the years 2003- 2004, 2002-2003 and 2001- 2002. Where as the consolidated financial information of the group for the years 2002-03 and 2003-04 are based on audited accounts for those years, information for the year 2001-02 is based upon the audited accounts regrouped / rearranged / reclassified, wherever considered necessary, while furnishing previous years figures for the year 2002-03. The consolidated financial statements were audited and reported upon by the respective auditors, names of whom and the period of their audit are given below: Year Name of Auditors 2001-02 M/s SBA Associates, GP Agrawal & Co., Parakh & Co., Kalani & Co., Surendar K. Jain & Co., Mookherjee Biswas & Pathak 2002-03 M/s Parakh & Co., Kalani & Co., Surendar K. Jain & Co, Mookherjee Biswas & Pathak, M.C.Bhandari & Co, P.K.Chopra & Co 2003-04 M/s Kalani & Co, Surendar K Jain & Co, Mookherjee Biswas & Pathak, M C. Bhandari & Co, P.K.Chopra & Co, G.P.Kapadia & Co The Bank did not prepare consolidated financial statements for the years 1999-2000 and 2000-2001 since the relevant accounting standard was not mandatory for those years and hence the same are not set out in this report. The bank also has not prepared consolidated financial statements for the six months ended 30th September 2004. 15. Annexure H is as per the consolidated financial statements of the Bank for the respective years as detailed in paragraph 14 above. We have not carried out any review, audit or adjustments to the same. However we have compared the financial information in annexure H with the Bank’s consolidated financial statements as detailed in paragraph 14 above. 16. Further we have examined the following financial information relating to the Bank on a standalone basis, proposed to be included in the offer documents, as prepared and approved by the Bank and annexed to this report. (i) Summary of accounting ratios based on the adjusted profits relating to earnings per share, net asset value and return on net worth enclosed as annexure I (ii) Capitalization statement as at 31st March 2004 of the Bank enclosed as annexure J F - 4 (iii) Statement of tax shelters enclosed as annexure K (iv) Statement of borrowings enclosed as annexure L All the above data relate to the bank as a whole on a stand-alone basis. 17. a) In our opinion, the financial information of the Bank as stated in annexure A above read with respective significant accounting policies, after making groupings and adjustments as were considered appropriate by us and subject to non-adjustment of certain matters as stated in the said annexure, have been prepared in accordance with the SEBI Guidelines. b) In our opinion, the financial information as given in annexures B to L have been properly extracted from Bank’s audited financial statements, unaudited financial statements as prepared by the bank for the half years ended 30th September 2003 and 2004 or have been correctly prepared from the financial information in annexure A as applicable and in all cases are in accordance with the SEBI guidelines. 18. This report is intended solely for your information and for inclusion in the offer document in connection with the public issue of the shares of the Bank and is not to be used, referred to or distributed for any other purpose without our prior written consent. 19 This report should neither in any way be construed as a reissuance or redrafting of any of the previous audit reports issued by us or by other firms of chartered accountants nor construed as a new opinion on any financial statements referred to herein. 20 This is in super cessation of our report dated 10th January 2005. For Surendar K Jain & Co For Mookherjee Biswas & Pathak Chartered Accountants Chartered Accountants Sanjeev Chaudhary S. K. Pathak (Partner) (Partner) Membership No 85761 Membership No 2480 For M C Bhandari & Co For P K Chopra & Co Chartered Accountants Chartered Accountants M R Jain Arvind Mongia (Partner) (Partner) Membership No 50919 Membership No 85176 For Ramanlal G Shah & Co For B K Ramadhyani & Co Chartered Accountants Chartered Accountants Vivek S Shah Shyam Ramadhyani (Partner) (Partner) Membership No 112269 Membership No 19522 F - 5 Annexure A I SUMMARY STATEMENT OF PROFIT AND LOSS AS RESTATED (Rs. in Million) S. AUDITED LIMITED REVIEW NO. FINANCIAL YEARS ENDED 31ST MARCH HALF YEAR 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 A INCOME 1 Interest Earned 51,545.5 58,634.8 66,478.7 74,850.1 77,797.0 38,168.9 41,431.2 1.1 Interest & Discount on advances/bills 25,142.3 28,235.4 33,178.8 37,115.9 38,760.1 19,319.9 20,732.4 1.2 Income on Investment 24,171.0 27,417.8 30,027.6 32,983.3 36,809.7 17,810.9 19,706.4 1.3 lnterest on balance with RBI and other Inter Bank Lending 1,715.4 1,522.9 2,276.3 1,793.8 1,131.3 561.9 656.1 1.4 Interest on Income Tax 168.6 896.8 163.8 2,152.7 191.8 8.4 76.2 1.5 Others 348.2 561.9 832.2 804.4 904.1 467.8 260.1 2 OTHER INCOME 7,276.6 7,784.2 9,777.2 12,503.1 18,668.8 10,490.3 10,503.3 2.1 Commission, Exchange & Brokerage 3,817.1 4,193.5 4,339.9 4,800.1 5,519.0 2,498.2 3,218.0 2.2 Profit on sale of investments (Net) 2,149.2 2,420.3 4,379.1 6,722.8 12,363.7 6,989.8 2,786.1 2.3 Profit on revaluation of investments (Net) -0.1 -257.5 -581.7 -700.8 -1,178.0 -7.2 -520.8 2.4 Profit on sale of land,bldg & other assets (Net) 6.8 3.7 3.7 4.1 2.4 1.4 3.8 2.5 Profit on exchange transaction ( Net) 761.6 941.5 922.4 950.8 1,060.2 556.7 669.5 2.6 Income earned by way of dividends etc. from subsidiaries/companies/ jt. Ventures in India 202.5 30.0 280.0 237.0 303.0 183.0 163.0 2.7 Amount transferred from interbranch transactions blocked account 3,870.7 2.8 Miscelleneous Income 339.5 452.7 433.8 489.1 598.5 268.4 313.0 TOTAL INCOME 58,822.1 66,419.0 76,255.9 87,353.2 96,465.8 48,659.2 51,934.5 F - 6 (Rs. in Million) S. AUDITED LIMITED REVIEW NO. FINANCIAL YEARS ENDED 31ST MARCH HALF YEAR 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 B EXPENDITURE 1 Interest Expended 35,382.0 38,250.5 43,525.8 43,612.9 41,549.9 20,858.2 22,379.7 1.1 Interest on deposits 33,667.9 36,094.6 41,216.2 41,625.6 39,264.4 19,744.6 21,006.4 1.2 Interest on RBI / Inter-Bank borrowings 290.7 392.3 478.9 75.3 133.4 34.7 174.1 1.3 Others 1,423.4 1,763.6 1,830.7 1,912.0 2,152.1 1,078.9 1,199.2 Operating expenses 15,238.5 18,716.4 17,992.1 20,567.3 23,707.3 10,703.5 13,197.5 1 Payment to & provision for employees 11,836.7 14,590.8 13,163.2 14,760.8 16,540.6 7,602.6 9,515.6 2 Rent, Taxes & Lighting 719.8 824.1 943.1 1,055.7 1,199.5 580.3 644.9 3 Printing and Stationery 223.4 244.5 285.5 525.1 350.9 169.2 178.9 4 Advertisement and Publcity 43.4 47.1 84.2 78.4 108.5 35.4 58.6 5 Depreciation on Banks Properties (net of amounts adjusted against revaluation reserve) 429.9 746.3 852.3 1,289.8 1,814.5 658.8 705.0 6 Director’s Fees, Allowances & Expenses 1.2 1.7 3.1 2.8 3.5 1.5 2.6 7 Auditors’ fees & Expenses 72.0 88.5 133.1 155.7 178.8 73.6 100.7 8 Law Charges 58.4 60.3 76.1 85.8 132.5 61.9 52.7 9 Postage, Telegrams, Telephones etc. 236.6 256.7 285.0 313.1 451.1 197.2 256.2 10 Repairs & Maintenance 136.8 169.7 189.8 199.2 244.2 115.6 136.0 11 Insurance 305.5 331.1 388.8 461.0 546.6 272.7 413.5 12 Other Expenditure 1,174.8 1,355.6 1,587.9 1,639.9 2,136.6 934.7 1,132.8 TOTAL EXPENDITURE 50,620.5 56,966.9 61,517.9 64,180.2 65,257.2 31,561.7 35,577.2 Gross Profit before provisions & contigencies 8,201.6 9,452.1 14,738.0 23,173.0 31,208.6 17,097.5 16,357.3 LESS: Provisions & Contingencies (other than floating provision for NPAs) 4,120.2 4,815.7 6,714.1 11,151.0 12,191.7 5,852.3 9,005.1 Profits after provisions and contingencies 4,081.4 4,636.4 8,023.9 12,022.0 19,016.9 11,245.2 7,352.2 Less: Floating provision for NPAs 0.0 0.0 2,400.0 3,600.0 7,930.0 5,730.0 0.0 Net Profit for the Year / half year As per Financial Statements 4,081.4 4,636.4 5,623.9 8,422.0 11,086.9 5,515.2 7,352.2 F - 7 (Rs. in Million) S. AUDITED LIMITED REVIEW NO. FINANCIAL YEARS ENDED 31ST MARCH HALF YEAR 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 Adjustments due to change in accounting policies/wage revision Adjustments on account of : (a) Changes in Accounting Policies Amortisation of Goodwill on account of amalgamation of eNBL 0.0 0.0 0.0 -373.3 373.3 186.7 0.0 (b) Impact of wage revision 0.0 0.0 0.0 -460.0 -1,104.0 -552.0 861.0 Total 0.0 0.0 0.0 -833.3 -730.7 -365.3 861.0 Tax impact of adjustments 0.0 0.0 0.0 306.2 254.9 127.5 -303.9 Total adjustments 0.0 0.0 0.0 -527.1 -475.8 -237.8 557.1 Total PROFIT 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 7,909.3 Transfer from Other Reserves 584.9 Total Adjusted Net Profit 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 8,494.2 APPROPRIATIONS TRANSFER TO Statutory Reserve 1,020.3 1,159.1 1,406.0 2,105.5 2,771.7 967.7 * Revenue & Other Reserve @ 2,457.5 2,886.4 1,405.7 2,841.5 1,970.1 2,903.0 * Transfer to Invest. Fluctuation Reserve 36.0 0.0 1,755.8 1,900.0 4,648.5 * Transfer to Capital Reserve - Investment (HTM) 0.0 6.1 419.7 0.4 23.6 ** Dividends to Central Government /Public 500.0 530.7 636.7 928.5 1,061.2 0.0 0.0 Dividend Tax 67.6 54.1 0.0 119.0 136.0 0.0 0.0 Balance carried over to balance Sheet 0.0 0.0 0.0 0.0 0.0 5,277.4 4,623.5 TOTAL 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 8,494.2 F - 8 (Rs. in Million) S. AUDITED LIMITED REVIEW NO. FINANCIAL YEARS ENDED 31ST MARCH HALF YEAR 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 ^^ Break-up of provisions and contingencies Provision for Non Performing Advances 1,974.6 3,258.0 3,913.3 4,729.7 4,010.4 2,405.2 1,182.1 Provision for Standard Accounts 515.0 147.0 150.0 140.0 465.0 50.0 130.0 Depreciation on Investments -77.9 200.8 -352.1 1,327.5 -308.6 -177.0 -429.0 Provisions for income tax 1,240.0 1,108.3 1,983.5 3,406.6 6,599.9 2,803.5 2,831.2 Provision for Restructured Standard Accounts 0.0 0.0 155.3 771.1 1,207.4 510.0 789.5 Others 468.5 101.6 864.1 776.1 217.6 260.6 4,501.3 4,120.2 4,815.7 6,714.1 11,151.0 12,191.7 5,852.3 9,005.1 Floating Provision for NPAs (in addition to norms) 0.0 0.0 2,400.0 3,600.0 7,930.0 5,730.0 0.0 Total 4,120.2 4,815.7 9,114.1 14,751.0 20,121.7 11,582.3 9,005.1 * As per convention, appropriation are generally carried out at the end of the financial year, except for Rs.967.7 million tranfered to Statutory Reserves and Rs.2903.0 million transfered to Revenue and Other Reserves, out of Blocked Accounts, as per RBI’s permission. ** In terms of the Bank’s accounting policies, profit on sale of investments in “Held to Maurity” category is initially credited to Profit & Loss Account and an equivalent amount is appropriated to “Capital Reserve Account” at the end of the year @ Revision in profits in the years 2002-03 and 2003-04 due to adjustments made have been notionally adjusted in ‘Transfer to revenue & other reserves. F - 9 II SUMMARY STATEMENT OF ASSETS AND LIABILITIES AS RESTATED (Rs. in Million) S. AUDITED LIMITED REVIEW NO. As at March 31 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 (A) ASSETS 1 Cash in Hand 4,281.3 4,452.0 5,777.7 6,288.1 6,992.1 5,659.8 5,570.0 Cheques/DDs in hand 0.0 0.0 1,532.5 0.0 0.0 0.0 0.0 2 Balance with RBI 50,492.7 49,204.2 43,704.1 59,397.1 60,430.7 41,429.6 60,846.4 3 Balance with Banks 7,525.8 7,017.3 11,150.7 14,322.4 13,811.9 9,978.8 14,107.6 - In India 1,313.8 2,813.1 5,992.6 13,547.8 12,816.3 5,899.3 13,804.8 - Outside India 6,212.0 4,204.2 5,158.1 774.6 995.6 4,079.5 302.8 4 Money at call and Short Notice 0.0 18.6 1,818.3 764.2 6,970.4 6,759.2 50.6 5 Investments - In India 220,903.1 251,197.2 281,970.5 340,206.7 421,161.3 417,339.6 460,092.2 - Outside India 87.6 87.0 101.2 93.8 93.6 1,929.7 93.6 TOTAL 220,990.7 251,284.2 282,071.7 340,300.5 421,254.9 419,269.3 460,185.8 6 Advances - In India 225,717.2 280,290.5 343,694.2 402,281.2 472,247.2 401,968.2 518,705.3 - Outside India 0.0 0.0 0.0 0.0 0.0 0.1 7 Fixed Assets 6,805.3 7,239.2 7,956.1 8,473.7 8,998.4 8,787.8 9,084.2 Less Revaluation Reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 Net Fixed Assets 3,253.1 3,772.5 4,574.8 5,177.9 5,788.0 5,534.7 5,916.6 8 Other Assets 25,472.8 35,545.1 31,441.3 30,323.7 33,172.9 32,554.1 32,300.5 TOTAL (A) 537,733.6 631,584.4 725,765.3 858,855.1 1,020,668.1 923,153.7 1,097,682.9 (B) LIABILITIES 1 DEPOSITS 474,832.3 561,311.3 641,234.8 758,135.0 879,163.9 801,848.9 945,886.4 Demand Deposits From Banks 1,518.1 1,477.2 1,112.5 1,660.2 1,431.8 1,438.1 929.9 Demand Deposits From others 52,851.9 61,636.7 66,471.6 97,222.7 97,572.0 81,190.0 96,552.2 2 Saving Deposits 158,754.1 185,300.6 216,642.1 256,478.9 304,226.1 279,108.6 330,151.0 3 Term Deposits From Banks 9,778.3 12,424.0 13,161.7 4,847.8 7,178.2 4,592.5 3,512.9 Term Deposits From others 251,929.9 300,472.8 343,846.9 397,925.4 468,755.8 435,519.7 514,740.4 4 Borrowings 6,624.3 6,732.0 4,085.7 6,621.6 12,890.6 7,452.5 19,520.2 - In India 6,561.3 6,661.0 4,041.7 5,608.9 6,260.0 4,153.8 6,405.1 - Outside India 63.0 71.0 44.0 1,012.7 6,630.6 3,298.7 13,115.1 5 Other liabilities and Provisions 28,605.6 31,417.3 38,220.7 41,662.9 59,130.2 53,505.5 55,033.6 6 Subordinate Debts 8,497.7 8,898.5 11,798.6 15,928.6 23,578.6 18,578.6 23,428.6 TOTAL (B) 518,559.9 608,359.1 695,339.8 822,348.1 974,763.3 881,385.5 1,043,868.8 F - 10 (Rs. in Million) S. AUDITED LIMITED REVIEW NO. As at March 31 30th Sept 30th Sept 2000 2001 2002 2003 2004 2003 2004 (C) NET ASSETS (C=A-B) 19,173.7 23,225.3 30,425.5 36,507.0 45,904.8 41,768.2 53,814.1 (D) Share Capital 2,122.4 2,122.4 2,122.4 2,653.0 2,653.0 2,653.0 2,653.0 (E) Share Application Money 0.0 0.0 1,644.9 0.0 0.0 0.0 0.0 (F) RESERVES AND SURPLUS 1 Statutory Reserve 4,468.1 5,627.2 7,033.2 9,138.7 11,910.4 9,138.7 12,878.1 2 Capital Reserve 130.5 136.6 556.2 556.6 580.3 556.6 580.3 3 Revaluation Reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 4 Investment Fluctuation Reserve 1,345.4 1,345.4 3,101.3 5,001.3 9,649.8 5,001.3 9,649.8 5 Revenue and other Reserve 11,107.3 13,993.7 15,399.3 17,474.9 19,428.8 17,458.7 21,746.9 6 Deferred Tax Reserve 0.0 0.0 568.2 568.2 568.2 568.2 568.2 7 Balance of Profit & Loss Account 0.0 0.0 0.0 0.0 0.0 5,277.4 4,623.5 8 Share Premium 0.0 0.0 0.0 1,114.3 1,114.3 1,114.3 1,114.3 TOTAL 20,603.5 24,569.6 30,039.5 37,149.8 46,462.2 42,368.3 54,328.7 Less Revaluation Reserve 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 TOTAL (F) 17,051.3 21,102.9 26,658.2 33,854.0 43,251.8 39,115.2 51,161.1 TOTAL (D+E+F) 19,173.7 23,225.3 30,425.5 36,507.0 45,904.8 41,768.2 53,814.1 (G) CONTINGENT LIABILITIES 1 Claims against the Bank not acknowledged as debt 2,727.5 2,507.4 2,464.1 1,811.5 2,146.8 2,041.5 2,130.8 2 Disputed income tax demand under appeal/ references etc. 6,557.6 3,338.6 3,120.5 141.9 377.5 141.9 375.7 3 Liability for partly paid investments 1.8 1.8 3.4 3.4 1.8 1.8 1.8 4 Liability on account of outstanding forward exchange contracts 39,420.4 126,187.3 164,314.5 163,640.3 195,434.7 182,276.2 224,095.9 5 Guarantees given on behalf of constituents In india 19,470.6 21,191.3 26,162.4 31,501.9 39,950.7 38,492.0 47,064.6 Outside India 6,131.2 4,307.7 4,494.2 3,823.5 11,314.5 9,433.3 12,383.2 6 Acceptances,Endorcements and other Obligations 19,777.6 24,409.0 28,074.5 33,764.3 71,679.4 42,009.7 90,870.3 7 Other items for which the bank is contingently liable 1,550.9 133.9 214.8 1,021.8 1,394.9 1,802.1 2,553.8 TOTAL (G) 95,637.6 182,077.0 228,848.4 235,708.6 322,300.3 276,198.5 379,476.1 Bills for collection 25,305.1 26,879.7 29,928.6 33,347.6 48,130.8 33,575.8 48,721.7 F - 11 III STATEMENT OF CASH FLOW AS RESTATED (Rs. in Million) S. AUDITED LIMITED REVIEW NO. YEARS ENDED 31ST MARCH HALF YEAR ENDED 2002 2003 2004 30.9.2003 30.9.2004 A. Cash Flow from Operating Activities (I) Net Profit after Tax 5,623.9 7,894.9 10,611.1 5,277.4 7,909.3 Add Provision for Tax (net of deferred tax) 1,983.5 3,100.4 6,345.0 2,676.0 3,135.1 Profit before taxes 7,607.4 10,995.3 16,956.1 7,953.4 11,044.4 (ii) Adjustment for : Depreciation Charges (Gross) 938.4 1,375.3 1,899.9 514.8 747.8 Less : Amount drawn from Revaluation Reserve (85.5) (85.5) (85.5) (42.7) (42.7) Provision for NPAs, Floating Provision towards NPAs 6,795.6 9,352.3 14,008.2 8,135.2 1,483.3 Provision on Standard Assets 605.3 1,211.1 1,672.4 560.0 919.5 Other Provisions (net) 576.2 306.1 589.3 812.7 3,339.2 Depreciation/ (Release) on Investments (net) (378.3) 1,497.4 (356.5) (177.1) (429.0) Dividend from Subsidiary/Others (Investing Activity) (280.0) (273.0) (303.0) 183.0 163.0 Interest on Subordinate debts (Financing Activity) 1,529.8 1,723.8 1,951.9 891.1 1,102.3 Amortisation Expenses/Deferred Revenue Expenses 1,139.5 1,523.6 767.4 575.4 575.1 Issue Expenses 54.2 0.0 0.0 0.0 (3.8) Profit / Loss on sale of Fixed Assets ( net ) (3.7) (3.4) (2.4) (1.4) 10,891.5 16,627.7 20,141.7 11,451.0 7,854.7 Operating Profit before Changes in Operating Assets andLiabilities 18,498.9 27,623.0 37,097.8 19,404.4 18,899.1 iii Adjustment for net change in Operating Assets and Liabilities Decrease / (Increase) in Investments (30,425.5) (59,726.1) (80,597.9) (78,960.6) (37,492.4) Decrease / (Increase) in Advances (70,058.1) (64,339.3) (76,046.0) (7,822.2) (47,640.3) Increase / (Decrease) in Deposits 79,923.5 116,900.2 121,029.0 43,714.0 66,722.4 Increase / (Decrease) in Borrowings (2,646.2) 2,535.9 6,268.9 830.8 6,629.6 Decrease / (Increase) in Other Assets 54.9 (1,898.2) (612.9) (5,991.6) (5,960.5) Increase / (Decrease) in Other Liabilities & Provisions (767.8) (2,851.9) 7,988.7 11,521.0 (7,817.4) (23,919.2) (9,379.4) (21,970.2) (36,708.6) (25,558.6) Cash generated from Operations (5,420.3) 18,243.6 15,127.6 (17,304.2) (6,659.5) Tax Paid (net of refund ) 2,288.3 (1,234.9) (9,834.7) 506.1 2,817.5 Net Cash from Operating Activities (3,132.0) 17,008.7 5,292.9 (16,798.1) (3,842.0) B. Cash Flow from Investing Activities Investment in Subsidiary Cos/ JV/RRBs (10.0) 0.0 0.0 168.9 (1,009.5) Purchase of Fixed Assets (net of Sales) (1,280.2) (2,262.6) (2,048.9) (827.5) (829.7) Dividend recd from Subsidiaries / JV / RRBs 280.0 273.0 303.0 (183.0) (163.0) Loss of PNB Caps taken over 0.0 0.0 (16.1) (16.1) Net Cash used in investing Activities (1,010.2) (1,989.6) (1,762.0) (857.7) (2,002.2) F - 12 (Rs. in Million) S. AUDITED LIMITED REVIEW NO. YEARS ENDED 31ST MARCH HALF YEAR ENDED 2002 2003 2004 30.9.2003 30.9.2004 C. Cash Flow from Financing Activities Subordinate Bonds Issued 4,800.0 4,150.0 7,650.0 2,650.0 0.0 Redemption of Subordinate Bonds (1,899.8) (20.0) 0.0 0.0 (150.0) Share Application Money 6,753.9 0.0 0.0 0.0 0.0 Issue Expenses (54.2) 0.0 0.0 0.0 0.0 Interest paid on Subordinate Debts (1,529.8) (1,723.8) (1,951.9) (891.2) (1,102.4) Payments of Dividends / Corporate tax on Dividend (636.7) (636.7) (1,795.8) (1,047.5) (533.9) Net Cash from Financing Activities 7,433.4 1,769.5 3,902.3 711.3 (1,786.3) D Net Change in Cash and Cash Equivalents 3,291.2 16,788.6 7,433.2 (16,944.5) (7,630.5) Cash and Cash Equivalents at the beginning of the year 60,692.1 63,983.3 80,771.9 80,771.9 88,205.1 Cash and Cash Equivalents at the end of the year 63,983.3 80,771.9 88,205.1 63,827.4 80,574.6 3,291.2 16,788.6 7,433.2 (16,944.5) (7,630.5) Note :- 1 All figures in bracket represent “Cash Outflow” 2 Direct taxes paid (net of refund) are treated as arising from operating activities and are not bifurcated between investing and financing activities. 3 Cash Flow Statements have not been furnished for the years 1999-2000 and 2000-2001, since accounting standard - 3 issued by the Institute of Chartered Accountants of India was not mandatory for the years. 4 Cash flows for the year 2001-02 are as previous years regrouped figures published for the year 2002-03 F - 13 IV SIGNIFICANT ACCOUNTING POLICIES as at 31ST MARCH 2004 1. ACCOUNTING CONVENTIONS The accompanying financial statements are prepared on historical cost basis and conform to the statutory provisions and prevailing practices, except as otherwise stated. 2. TRANSACTIONS INVOLVING FOREIGN EXCHANGE 2.1 Monetary assets and liabilities, guarantees, acceptances, endorsements and other obligations are translated in Indian Rupee equivalent at the exchange rates prevailing at the end of the year as per Foreign Exchange Dealers’ Association of India (FEDAI) guidelines except (i) foreign currency deposits under various schemes and equivalent currency investments and other balances which are stated at notional rates, and (ii) advances of erstwhile London branches which are accounted for at the exchange rate prevailing on the date of parking in India. 2.2 Non-monetary items other than fixed assets are translated at exchange rate prevailing on the date of transaction. 2.3 Forward exchange contracts are translated at the exchange rate prevailing on the date of commitment. Gain/loss on evaluation of outstanding forward exchange contracts is taken to revenue as per FEDAI guidelines. 2.4 Income and expenditure items are accounted for at the exchange rate prevailing on the date of transaction. 3. INVESTMENTS 3.1 Investments are classified into six categories as stipulated in the Form A of the third schedule to the Banking Regulation Act, 1949. 3.2 The Investments are categorised into “Held to Maturity”, “Available for Sale” and “Held for Trading” in terms of RBI guidelines. 3.3 a) The securities acquired by the Bank with the intention to hold till maturity, not exceeding 25% of total investments, are classified under “Held to Maturity”. The securities acquired by the Bank with the intention to trade by taking advantage of short-term price/ interest rates movements are classified under “Held for Trading”. The securities, which do not fall within the above two categories, are classified under “Available for Sale”. b) Transfer of securities from one category to another is carried out at least of acquisition cost/book value/market value on the date of transfer. The depreciation if any, on such transfer is fully provided for. 3.4 Investments are valued on the following basis: i) Held to Maturity: Valued at acquisition cost, unless more than the face/ maturity values, in which cases the premium is amortised over the remaining years to maturity. ii) Available for Sale: ii)&iii)Valued scrip wise and depreciation/ appreciation is aggregated classification wise in each category . While net depreciation is provided for, net appreciation is ignored. iii) Held for Trading: 3.5 Investments under “Available for Sale” and “Held for Trading” are valued as follows: a) Govt.Securities I. Central Govt. Securities At market price as per quotations put out by Fixed Income Money Market and Derivatives Association of India (FIMMDA) II. State Govt.Securities On appropriate yield to maturity basis as per FIMMDA guidelines b) Securities guaranteed by Central/ On appropriate yield to maturity basis as per FIMMDA guidelines State Government, PSU Bonds (not in the nature of advances) c) Treasury Bills At carrying cost d) Equity Shares At market price, if quoted, otherwise at Break up value of the Share as per latest Balance Sheet (not more than one year old), otherwise at Re.1 per company F - 14 e) Preference Shares At market price, if quoted, or on appropriate yield to maturity basis not exceeding redemption value as per FIMMDA guidelines f) Debentures At market price, if quoted, otherwise on appropriate yield to maturity basis (not in the nature of advances) as per FIMMDA guidelines g) Mutual Fund As per stock exchange quotation, if quoted; at repurchase price/NAV, if unquoted h) Commercial Papers At carrying cost i) Investment in sponsored At carrying cost Regional Rural Banks j) Investment in subsidiaries At carrying cost less diminution and joint ventures k) Other Investments At carrying cost less diminution 3.6 Investments are subject to appropriate provisioning/ de-recognition of income, in line with the prudential norms of NPA classification. Debentures/Bonds in the nature of advances are subjected to usual prudential norms. 3.7 Profit/ Loss on sale of Investments in any category is taken to Profit and Loss account. However, in case of profit on sale of investments in “Held to Maturity” category, an equivalent amount is appropriated to “Capital Reserve Account”. 3.8 Securities repurchased/resold under buy back arrangement are accounted at original cost. 3.9 As per RBI guidelines the different category of swaps are valued as under: Hedge Swaps Interest rate swaps which hedges interest bearing asset or liability are accounted for on accrual basis except the Swap designated with an asset or liability that is carried at market value or lower of cost or market value in the financial statement. Gain or losses on the termination of swaps are recognised over the shorter of the remaining contractual life of the swap or the remaining life of the asset/liability. Trading Swaps Trading swap transactions are marked to market with changes recorded in the financial statements. 3.10 Incentive/ Front-end fees etc. received on subscription to securities are deducted from the cost of securities. 4. ADVANCES 4.1 Advances are classified as performing and non-performing assets and provisions are made in accordance with prudential norms prescribed by RBI. 4.2 Advances are stated net of provisions and de-recognised/ suspended interest relating to non-performing assets. 5 FIXED ASSETS 5.1 Premises including freehold/leasehold land and capital work-in-progress and other fixed assets are stated at historical cost except such premises, which have been revalued. The appreciation on revaluation is credited to Revaluation Reserve and depreciation provided thereon is deducted there from. 5.2 (a) Depreciation on assets (including land where value is not separable) is provided on straight-line method based on estimated life of the asset. Premises, including Land & Buildings, acquired on leasehold basis are amortised over the lease period. In respect of assets taken over from erstwhile New Bank of India and Nedungadi Bank Ltd., the estimated life has been determined in broad groups/ categories instead of individual asset. (b) Depreciation on additions to assets is provided for full year irrespective of date of acquisition and no depreciation is provided on assets sold/disposed off during the year. 5.3 Cost of acquisition of Computer Software Systems is amortised over a period of its economic life, maximum upto 5 years, except software costing upto Rs.5000/-, is charged to revenue. F - 15 6. STAFF BENEFITS Expenses on Gratuity, Pension and Leave encashment are accounted for on the basis of actuarial valuation. 7. AMORTISATION OF VRS EXPENDITURE Payments under Voluntary Retirement Scheme are amortised over a period of 5 years as per RBI guidelines. 8. REVENUE RECOGNITION 8.1 Income/expenditure is generally accounted for on accrual basis. 8.2 Income on non-performing assets is recognised on realisation as per RBI guidelines. 8.3 Recovery in non-performing advances is appropriated first towards interest including de-recognised/ suspended interest and recorded interest and thereafter towards (i) arrears of instalment in term loans and (ii) principal irregularity in other accounts. However, recovery in suit filed, decreed accounts and compromise cases is first appropriated towards principal or as per terms of decree/settlement. 8.4 Commission, interest on overdue bills, exchange, locker rent, income from merchant banking transactions and dividend income on mutual fund are accounted for on realisation. 8.5 Income from interest on refund of income tax and interest tax is accounted for in the year the order is passed by the concerned authority. 9. TAXES ON INCOME Taxes on income for the year comprises of current tax liability and deferred tax which recognises, subject to the consideration of prudence in respect of deferred tax assets, timing differences, being the difference between taxable income and accounting income that originate in one period and are capable of reversal in one or more subsequent periods. 10. OTHERS 10.1 Interest on matured term deposits for overdue period is accounted for as and when such deposits are renewed, as per Bank’s rules. 10.2 Gain/loss on sale of shares is accounted for net of brokerage. V. NOTES TO ADJUSTMENTS CARRIED OUT: a) As per significant accounting policies declared for the year 2002-03, goodwill arising out of amalgamation of erstwhile Nedungadi Bank Ltd. (eNBL) with the Bank was being amortized over a period of four years. Due to change in the accounting policy in the year 2003-04, the balance amount has been charged in that year itself. b) Pending settlement of wage revision of employees, as a prudent measure, an adhoc provision of Rs. 1,590 million (debited to provisions and contingencies) had been made by the bank during the half year ended 30th September 2004. Subsequently, the Indian Banks Association had announced that an understanding had been reached with the Workmen unions and Officers’ association. However, we have been informed by the bank that a memorandum of understanding is yet to be executed in this respect and that several matters, which would in turn materially affect the quantification of liability upto 30th September 2004, are yet to be finalized. However, based on the bank’s best estimate of liability, an additional provision of Rs.703 million is considered necessary. Year wise estimated liability as made by the bank is as under: - Rs. In million Year Managements best Provision made in books Difference adjusted estimate of Liability in restated Profit & Loss Account 2002-03 460 -460 2003-04 1,104 -1,104 Half year ended 30/9/2004 729 1,590 861 Half year ended 30/9/2003 552 -552 Consequential adjustments have been made. F - 16 VI. ADJUSTMENTS NOT CARRIED OUT IN THE STATEMENT OF PROFIT & LOSS AND ASSETS & LIABILITIES: A. Accounting Policies 1. During the year 2002-03, bank had changed the method of accounting of leave encashment from cash to accrual method based on actuarial valuation. Since actuarial valuation for earlier years was not available, the effect could not be quantified. The unprovided liability relating to earlier years of Rs.765.9 million was debited to ‘Other Reserves” in the year 2002-03. 2. During the five years period ended March 31, 2004 and half-year ended 30TH Sept 2004 Reserve Bank of India (RBI) has issued various guidelines on income recognition, asset classification and provisioning in respect of non-performing advances and valuation of/ depreciation on investments. The Bank has carried out necessary amendments in its accounting policies in the relevant years to be in conformity with the said RBI guidelines. Accordingly, the amounts for respective years are based on RBI guidelines prevailing in the respective years. 3. From the year 2001-02, cost of acquisition of computer software systems is amortized over a period of its economic life, up to a maximum of 5 years. However, from the year 2002-03, software costing up to Rs.5000/ is charged to revenue. In earlier years i.e. prior to 2001-02, software expenditure was charged to revenue in the year of purchase. The effect of the same could not be quantified and adjusted. 4. From 01st April 2002, guarantees, acceptances, endorsements and other obligations in foreign currency are translated in Indian rupees equivalent at the exchange rates prevailing at the end of the year as per Foreign Exchange Dealers’ Association of India (FEDAI). Prior to this these were translated at the exchange rate prevailing on the date of commitment. B. Auditors’ Report 1. Unrealized gain of Rs.76.8 million arising out of revaluation of Investments under “HFT category” ’was recognized in the year 2000-01. Since the same had been carried out as per RBI guidelines, effect has not been given in the aforesaid restated financial information. C. Amalgamation of PNB Capital Services Limited (appointed day 1.4.2003) and Nedungadi Bank Limited (appointed day 1.2.2003) 1. The working results of these companies for the periods prior to their respective appointed day have not been adjusted in the restated Profit and Loss Account. D. Others 1. Provisions for tax expense for each of the five years have been adopted from their respective financial statement. For want of necessary information, they have not been recast depending on assessed liabilities taking into account appellate orders. 2. The bank is in the process of identifying impaired assets, if any as required by Accounting Standard 28 issued by the Institute of Chartered Accountants of India. No effect has been given in the accounts for adjustments required if any. However a provision of Rs.50 million has been made on an adhoc basis. F - 17 VII MATERIAL NOTES ON ACCOUNTS YEAR ENDED 31ST MARCH 2000 1. The liability towards pension fund determined on the basis of actuarial valuation has been contributed in full save and except that in respect of employees retired up to 31st March 1998, contribution has been made to the extent of Rs.1517.7 million which is inclusive of Rs.550.0 million contributed during the year as against actuarial valuation of such liability at Rs. 1907.7 million. Balance liability of Rs.390.0 million to be contributed by the year ending 31ST March 2001. 2. Consequent upon change in the procedure of calculation of interest on term deposits (multi benefit scheme) on completed quarter basis as against anniversary date basis followed previously, at a number of branches, interest pertaining to earlier years is accounted for during the year, the quantum of which could not be determined in view of large number of account involved. YEAR ENDED 31ST MARCH 2001 1. In accordance with RBI guidelines, securities in HFT category have been revalued and a sum of Rs.76.8 million remaining unrealized has been taken to profit resulting in increase in profit by an equivalent amount 2. Other Assets include a sum of Rs. 122.1 million recovered by State Bank of India out of TT remittances, towards interest on wrong/ double payments of TTs, which has been disputed by the Bank and hence no provision has been made. This was expensed out in a subsequent year on finalization of the matter. YEAR ENDED 31ST MARCH 2002 1. In accordance with principles enunciated in AS-26 “Intangible Assets”, the Bank changed accounting policy in respect of acquisition of software system, the cost of acquisition whereof has been treated as Intangible Assets, to be amortized over the economic life of such assets, maximum up to 5 years. Up to the previous year, such expenses were charged to revenue. Had the bank continued the earlier policy, the profit for the year would have been lower by Rs 371.6 million. 2. Leave encashment was accounted for on cash basis. However the bank has made an adhoc provision of Rs 250.0 million during the year in this respect. 3. a) During the year, the Bank has come out with a public issue of Rs. 1,644.9 million comprising of 53,060,700 fully paid up equity shares of Rs 10/- each at a premium of Rs 21/-. The issue opened on 21st March 2002 and was closed on 28th March 2002. Out of subscription of Rs 6,964.3 million, a sum of Rs 1,644.9 million being the amount to the extent of issue size has been shown as “Share application money” in Balance Sheet. The balance amount net of stock Invest, over and above the issue size has been included in “Other Liabilities & Provisions - Others”. b) The issue expenses Rs 54.2 million incurred during the year have been charged to Profit & Loss Account. YEAR ENDED 31ST MARCH 2003 1. The bank has approved the restructuring of the Investment in IFCI, carried in “Available for Sale” category. The same involved rollover of the principal, conversion of the interest not due on bonds including zero coupon bonds, reduction of coupon rate for preference shares and bonds, effective 1st April 2002. In terms of permission granted by the Reserve Bank of India for the year, the post restructuring exposure to IFCI, has been shifted to “ Held to maturity” category at book value. As per policy consistently followed by the bank for transfer of securities, the difference between the market value and book value amounting to Rs.1,341.9 million has been provided for, though the bank had obtained permission from Reserve Bank of India that the restructuring and transfer of IFCI bonds to ‘ Held to maturity’ category will not attract the prudential norms. 2. The erstwhile Nedungadi Bank Ltd. (eNBL) was amalgamated with the Bank as per the scheme vide Government of India, Ministry of Finance & Company Affairs, Department of Banking Affairs (Banking Division) notifications no. 15/21/2002- BOA (i) & (ii) dated 31st January 2003 on the prescribed date i.e. 1st February 2003. As per scheme the business, properties, assets and liabilities of eNBL stand transferred to the Bank. F - 18 In terms of the scheme of amalgamation, the Bank undertook due diligence exercise for valuation of assets and determination of liabilities of eNBL. The details of assets & liabilities taken over are as under: ASSETS Amount (Rs.’000) LIABILITIES Amount (Rs.’000) Cash & Balance with RBI 761,749 Deposits 13,561,516 Balance with Bank, Money at call & short notice 493,243 Borrowings 9,644 Investments 5,696,019 Other Liabilities & Provisions 780,785 Advances 6,257,207 TOTAL 14,351,945 Fixed Assets 317,000 Excess of Liabilities over the Assets being Cost Other Assets 329,045 of business taken as Goodwill 497,682 TOTAL 13,854,263 In terms of the scheme, the advances including Bills purchased and discounted are classified into two categories (i) “Advances considered good and readily realizable”, included in Advances and (ii) “Advances considered not readily realizable and/ or bad or doubtful of recovery” taken on collection basis. The recovery in collection accounts will be appropriated in terms of the scheme. The accounting treatment for the amalgamation is given on the basis of purchase method. The asset classification, income recognition and provisioning norms in respect of advances categorized as ‘Advances considered good and readily realizable have been applied on ongoing basis. Depreciation on fixed assets acquired from eNBL is provided for the period, such assets are held by the bank. 3. During the year, bank has changed the accounting of leave encashment from cash to accrual method. The liability ascertained on the basis of actuarial valuation amounting to Rs.765.9 million (net of Rs.250 million provided in previous year and Rs.51.9 million provision of eNBL) pertaining to the year up to 31st March 2002 has been charged to Revenue and Other Reserve. Liability of Rs239.2 million for the current year been charged to revenue. Had the bank continued the earlier policy, profit for the year would have been higher by Rs.239.2 million. YEAR ENDED 31ST MARCH 2004 1. In terms of scheme of amalgamation, approved by the Board in its meeting held on 1st March 2003 and sanctioned by the Hon’ble High Court of Delhi vide order-dated 25th August 2003, PNB Capital Services Ltd, a wholly owned subsidiary of the Bank was merged with the Bank w.e.f. the appointed date 1st April 2003. The amalgamation has been accounted for under “Pooling of Interest Method” as prescribed in AS-14.The accumulated losses amounting Rs.16.1 million (net of Reserves) have been debited to Reserves (Revenue & Other Reserves). 2. a) Premises consisting of Land & Buildings held and capitalized prior to 31st March 1994 were revalued as on 31st March 1995 and such revaluation by the approved valuers resulted in an appreciation in the value of said land & buildings by Rs.4,007.0 million, which was credited to Revaluation reserve account – capital reserve. b) Incremental depreciation attributable to revaluation cumulative upto 31st March 2004 Rs.796.6 million has been set off against the revaluation reserve. 3. Other assets as at 31st March 2004 includes expenditure on VRS of Rs.1,160.8 Million to be amortized in subsequent periods / years. 4. Pending registration of title deeds, properties amounting to Rs.309.6 million are included in premises. 5. The inter branch reconciliation system prevalent in the bank prior to October 1996 was not able to provide details of outstanding credit entries. However, as per RBI guidelines, the bank worked out the amount of such credit entries for more than 5 years on the basis of available records as on 31st March 2004 and got the same verified by a firm of Chartered Accountants. Taking the same as base, an amount of Rs. 4,589.7 million (net of adjustments since carried out) has been included under ‘other liabilities’. 6 Profits earned by the bank under the Government debt buy back program.. Rs 2,930.1 million. 7 Profit on securities, which were categorized as Held to Maturity and sold under the said Government’s debt buy back program of Rs 1408.8 million was not been transferred to capital reserve in terms of clarification from the Reserve Bank of India. F - 19 HALF YEAR ENDED 30TH SEPTEMBER 2004 1 The Bank has provided an additional amount of Rs 1,230 million by debiting Profit and Loss Account towards pension and gratuity during the period. 2 Consequent upon transfer of certain government securities from available for sale AFS) to held to maturity (HTM) category in terms of Reserve Bank of India (RBI) circular number BP.BC.37/21.04.141/2004-05, depreciation amounting to Rs 2,077 million has been charged to Profit and Loss Account. 3 In terms of RBI’ s approval, an amount of Rs 3,870.7 million has been transferred to Profit and Loss Account from inter branch transactions blocked account and thereafter appropriated to statutory reserve (Rs 967.7 million) and revenue and other reserves (Rs. 2,903 million). Based on expert opinion, no provision for tax on such credit has been considered. HALF YEAR ENDED 30TH SEPTEMBER 2003 1 In terms of the scheme of amalgamation sanctioned by the honorable high court of Delhi vide order-dated 25.8.2003, PNB Capital Services Limited, a wholly owned subsidiary of the bank was merged with the bank with effect from 1.4.2003. Disclosure in terms of RBI guidelines: 31st March 2004 i) Share holding of Govt. of India - 80% ii) Lending to Sensitive Sectors: (Rs. in million) a) Capital Market Sector 145.4 b) Real Estate Sector 4,027.8 c) Commodity Sector 4,205.8 (Rs. in million) iii) Movement of NPAs: 2003-04 Gross NPAs Net NPAs* Opening Balance at the beginning of the year 49,800.6 15,269.1 Reduction during the year 13,543.8 16,238.8 Additions during the year 10,444.5 5,459.3 Closing Balance at the end of the year 46,701.3 4,489.6 including floating provision of Rs. 793 million for NPA iv) Maturity Pattern (Rs. in million) Maturity Loans & Investment Deposits Borrowings Foreign Foreign Pattern Advances in Securities currency currency Assets Liabilities 1-14 days 43,225.5 350.0 42,976.6 162.4 8,768.4 893.3 15-28 days 11,066.1 5,096.2 8,789.4 1,161.7 1,482.8 163.9 29 days to 3 months 33,136.7 6,020.0 34,844.5 3,657.6 6,059.0 485.0 Over 3 Months to 6 months 53,186.3 2,079.4 44,341.9 1,907.9 7,084.3 682.7 Over 6 Months to 12 months 65,511.0 3,759.5 71,343.2 1,305.2 1,285.6 1,760.7 Over 1 Year to 3 Years 145,361.0 34,073.0 451,566.5 4,673.5 584.2 4,459.4 Over 3 Years to 5 Years 54,331.9 55,063.0 24,529.9 17.3 158.7 93.4 Over 5 Years 66.428.7 314,813.8 200,771.9 5.0 41.2 88.4 Total 472,247.2 421,254.9 879,163.9 12,890.6 25,464.2 8,626.8 F - 20 (Rs. in million) v) Restructuring undertaken during: 2003-04 Total amount of Loan assets subjected to restructuring 4,197.0 The amount of Standard assets subjected to restructuring 3,741.9 The amount of Sub-Standard assets subjected to restructuring 427.2 The amount of Doubtful assets subjected to restructuring 27.9 (Rs. in million) vi) Corporate Debt Restructuring undertaken during: 2003-04 Total amount of Loan assets subjected to restructuring 9,163.5 The amount of Standard assets subjected to restructuring 7,294.8 The amount of Sub-Standard assets subjected to restructuring 318.8 The amount of Doubtful assets subjected to restructuring 1,549.9 vii) Bank’s net funded exposure for risk category-wise country exposures for each country is less than 2% of bank’s assets as on 31st March 2004 and as such no provision is required in terms of RBI Cir. No. DBOD.BP.BC.71/21.04.103/ 2002-2003 dated 19th February 2003 viii) Movement of Provisions (Rs. in million) (a) Provisions for NPAs (excluding provisions on standard assets)2003-04 Opening balance 28,353.3 Add: Provisions made during the year 14,016.5 Less: Write off/ write back during the year 5,848.1 Closing balance 36,521.7 {including floating provision of Rs.7,930 million for NPAs} (b) Provisions for depreciation on Investments: Opening balance 2,170.2 Add: Provisions taken over from PNB Capital Services Ltd. on amalgamation: 125.3 Add: Provisions made during the year 129.1 Less: Write off/write back of excess provisions during the year 437.7 Closing balance 1,986.9 ix) Details of financial assets sold during the year to Securitisation or Reconstruction Company (SC/RC) for asset reconstruction: (Rs. in million) i) No. of accounts: 3 ii) Aggregate value (net of provisions) of accounts sold to SC/RC 196.4 iii) Aggregate consideration 148.6 v) Aggregate Gain/Loss over net book value: (-) 47.8 F - 21 xii) Other Disclosures relating to Investments: a. Non Performing Non-SLR Investments (Rs. in million) GROUP-A Investments Opening Balance as at 01st April 2003 50.0 Additions during the year ended 31st March 2004 (identified during the year) 714.0 Reduction during the above period NIL Closing Balance as at 31st March 2004 764.0 Total Depreciation held 388.6 GROUP-B- Investments matured & Receivable-Parked under “Other Assets” Opening Balance as at 1st April 2003 333.7 Additions during the year ended 31st March 2004 89.1 Reduction during the above period 75.6 Closing Balance as at 31st March 2004 347.2 Total Provisions held 347.2 b. Repo/ Reverse Repo (Rs. in million) Minimum Maximum Daily As on outstanding outstanding average 31.03.04 during the during the outstanding year year during the year Securities purchased under reverse repos 2,000.0 51,000.0 15,051.6 NIL c. Derivatives (Rs. in million) HEDGE SWAPS a. Notional principal of Swap Agreements 15,454.0 b. Nature & Terms of the swaps including information on credit and market risk Interest rate swaps for hedging Tier II Bonds & Back to Back Swaps c. Losses which could be incurred if counter parties failed to fulfill their obligations 6.9 d. Collateral required by the entity upon entering into swaps NIL e. Whether any concentration of credit risk arising from the swaps NIL f. Fair value of the total swaps book (Accrual basis for hedge swaps) 10.2 F - 22 d. Issuer composition of Non-SLR investments (Rs. in million) Issuer Amount Extent Extent Extent of Extent of of private of ‘below ’unrated’ ’unlisted’ placement investment securities securities grade’ securities 1. PSUs 35,934.0 26,354.5 1,301.4 1,637.1 15,523.0 2. Fis 12,224.6 10,486.3 510.9 1,033.9 6,488.6 3. Banks 4,500.0 4,500.0 NIL 1,200.6 2,795.2 4. Private Corporates 6,858.6 6,700.0 NIL 547.6 3,008.2 5. Subsidiaries/joint ventures 2,064.6 NA NA NA NA 6. Others** 13,702.7 1,281.7 NA 149.5 492.5 7. Provision held towards depreciation-Non SLR (-)196.55 XXX XXX XXX XXX Total: 73,319.0 49,322.5 1,812.3 4,568.7 28,307.5 Excludes Rs.66.5 million relating to RRBs’ approved securities included in Subsidiary/JV category Includes Recapitalisation Bonds included in “Govt.Securities” amounting to Rs.6,250.7 Million. e. In accordance with RBI guidelines, the Bank’s Investment portfolio has been classified into three categories. The position of holding as on 31st March 2004 was as follows: Sl.No. Particulars % i) Held to Maturity (inclusive of 2.02% in exempted category) 25.81 ii) Available for Sale 72.80 iii) Held for trading 01.39 f. Investment in equity shares and equity like instruments outstanding is as under: (Rs. in million) Description As at 31st March 2004 i) Equity shares including Investments in subsidiaries and joint ventures 3,307.3 ii) Units of equity oriented Mutual Funds 1,253.6 iii) Regional Rural Banks 778.1 iv) Venture Capital Fund 225.0 Total 5,564.0 g. The position of profit booked on sale of the securities categorized in “Held to maturity” category is given hereunder: Security Sale Date Profit (Rs. in Million) GOI- 10.45 22nd July 2003 304.3 GOI-11.50 22nd July 2003 609.8 GOI-10.47 22nd July 2003 494.7 PRVUNL-11.70 12th August 2003 10.9 PRVUNL-11.70 4th September 2003 12.7 Total 1432.4 Out of above the profit on securities sold under Government’s Debt Buyback programme was Rs.1,408.8 million not being transferred to Capital Reserve in terms of clarification received from RBI. Balance amount of Rs.23.6 million, been appropriated to capital reserve F - 23 VIII. RELATED PARTY TRANSACTIONS: A. Key Management Personnel: i) Shri S.S. Kohli Chairman & Managing Director ii) Shri T.S. Narayanasami Executive Director Item Amount Amount Amount (Rs. in million) (Rs. in million) (Rs. in million) (2001-02) (2002-03) (2003-04) Remuneration 0.9 1.0 1.1 B. Name of associates Assets Care Enterprises Limited Everest Bank Limited Details of transactions: Rs. in million Type of Transaction FY 2002 FY 2003 FY 2004 Capital Contributed 15.0 13.0 - Dividend received - 7.9 7.9 Technical Services fees received 2.0 2.0 2.0 F - 24 IX. Segment Reporting A. For the year ended 31st March: (Rs. in million) Business Treasury Other banking Unallocated TOTAL Segments operations Particulars 2004 2003 2002 2004 2003 2002 2004 2003 2002 2004 2003 2002 Segment Revenue (external revenue) 49,138.7 40,638.0 35,744.2 47,327.1 46,715.1 40,511.7 XXX XXX XXX 96,465.8 87,353.1 76,255.9 Segment Results (before Prov & Contingencies) 19,123.0 12,192.7 7,630.3 15,019.4 12,869.2 8,447.6 XXX XXX XXX 34,142.4 25,061.9 16,077.9 Provisions & Contingencies (328.3) 1,504.7 (176.8) 13,842.1 9,839.6 7,300.5 1,104.0 460.0 XXX 14,617.8 11,804.3 7,123.7 Segment Results after Prov & Contingencies- before tax 19,451.3 10,688.0 7,807.1 1,177.3 3,029.6 1,147.1 (1,104.0) (460.0) XXX 19,524.6 13,259.6 8,954.2 Unallocated Corporate Expenses XXX XXX XXX XXX XXX XXX 2,568.5 2,262.3 1,339.0 2,568.5 2,262.3 1,339.9 Income tax XXX XXX XXX XXX XXX XXX XXX XXX XXX 6,345.0 3,100.4 1,990.4 Net Profit XXX XXX XXX XXX XXX XXX XXX XXX XXX 10,611.1 7,894.9 5,623.9 OTHER INFORMATION Segment Assets 433,726.8 353,463.5 293,354.5 582,112.1 498,450.5 428,092.5 8,039.6 10,236.9 7,776.4 1,023,878.5 862,150.9 729,223.4 Segment Liabilities 412,609.2 321,130.2 285,223.4 553,769.9 495,486.4 400,262.3 8,384.2 5,731.5 9,930.9 974,763.3 822,348.1 695,416.6 Capital Expenditure incurred during the year 23.3 4.5 3.7 1,700.7 1,526.5 1,160.7 286.3 190.0 129.8 2,010.3 1,721.0 1,294.2 Depreciation and amortisation 8.8 213.6 2.0 1,144.8 870.8 818.2 287.6 454.2 32.1 1,441.2 1,538.6 852.3 * Includes increase in asset value due to revaluation. F - 25 B) For the period ended 30th September 2004: (Rs. In million) Business Segments Treasury Other banking Unallocated Total operations Particulars Segment Revenue (external revenue) 22,383.4 29,551.1 XXX 51,934.5 Segment Results (before Prov & Contingencies) 5,823.3 11,898.9 XXX 17,722.2 Provisions & Contingencies 5,312.9 5,312.9 Segment Results after Provisions & contingencies 5,823.3 11,898.9 (5,312.9) 12,409.30 Unallocated Corporate Expenses XXX XXX 1,364.90 1,364.9 Income tax XXX XXX XXX 3,135.1 Net Profit XXX XXX XXX 7,909.3 Note: 1. The Bank does not have any secondary (geographical) segment. 2. Assumptions made by the management for segmental disclosure are not verifiable. Above date are as certified by the management. 3. Segmental information for the years ended 31st March 2000 and 31st March 2001 were not prepared by the bank. 4. The Bank has not computed segment assets, liabilities, capital expenditure incurred for the relevant period. 5. Provision for wage arrears has been treated as unallocated, pending apportionment of liability between two segments. F - 26 ANNEXURE B STATEMENT OF DIVIDENDS DECLARED BY THE BANK Year Ended Equity Capital No. of share Rate of Amount of (Rs.in Million) in million Dividend % dividend Rs. In Million 31.3.2000 2122.4 * 23.56 500.0 31.3.2001 2122.4 * 25.00 530.7 31.3.2002 2122.4 212.2 30.00 636.7 31.3.2003 2653.0 265.3 35.00 928.5 31.3.2004 2653.0 265.3 40.00 1061.2 * Consolidated capital not bifurcated in number of shares. F - 27 ANNEXURE C Phones: 23276964 23267547 Fax: 23267547 E-mail: rajkagg@yahoo.com RAJ K.AGGARWAL & ASSOCIATES CHARTERED ACCOUNTANTDS 3072/41, Gola market Near Golcha Cinema Darya Ganj New Delhi-110 002 The Managing Director, PNB Gilts Ltd., 5, Sansad Marg New Delhi Sub: Authentication of Information of PNB Gilts Ltd. We have reviewed the accompanying statements of “Summarized Profit and Loss Account” and “Summarized Statements of Assets and Liabilities” of PNB Gilts Ltd. for the Five and half years’ period ending 30th September 2004. Further. We have also reviewed the changes in accounting policies and notes to accounts for the said period on the basis of the records produced before us. FOR M/S RAJ. K. AGGARWAL & ASSOCIATES CHARTERED ACCOUNTANTS DATE : 10th January 2005 Sd/- PLACE : New Delhi PARTNER F- 28 PNB GILTS LIMITED SUMMARIZED PROFIT AND LOSS ACCOUNT (Rs. in millions) For the Year Ended March 31 2000 2001 2002 2003 2004 Half year ended 30.09.2004 INCOME Discount on: - Certificate of Deposits 1.2 0.9 0.0 0.0 12.1 31.1 - Commercial Paper 1.2 0.8 0.5 0.0 9.3 21.2 - Bills rediscounted 0.0 0.0 0.0 0.0 0.0 0.0 - Treasury Bills 171.2 77.9 122.4 67.4 83.0 151.0 173.6 79.6 122.9 67.4 104.4 203.3 Interest on: - Call & Notice Money 101.8 83.2 5.6 2.0 4.1 1.0 - Short-term 9.3 0.7 3.2 0.0 0.0 0.0 - Repo Lending 1.8 6.0 0.0 0.0 0.0 0.0 - Government Dated Securities 1,435.5 1,741.1 790.9 1,076.2 1,017.8 318.8 - PSU Bonds 86.6 93.7 123.7 119.5 92.7 28.3 1,635.0 1,924.7 923.4 1,197.7 1,114.6 348.1 Profit on: - Sale of Government Dated Securities 492.1 170.2 1,231.3 899.5 1,088.3 -827.6 - Sale of PSU Bonds 38.8 47.5 49.2 118.4 44.7 -36.8 530.9 217.7 1,280.5 1,017.9 1,133.0 -864.4 Other Income: 24.0 24.6 17.2 18.0 25.1 9.9 TOTAL INCOME 2,363.5 2,246.6 2,344.0 2,301.0 2,377.1 -303.1 EXPENDITURE Interest on: - Call & Notice Money 752.3 809.3 250.8 551.3 469.1 94.8 - Interest on Repo Borrowing 2.6 28.4 27.1 94.1 99.6 196.5 - Interest on RBI Borrowing 446.3 407.0 100.9 25.6 0.0 0.0 - Short Term Borrowing 59.2 100.9 127.7 35.7 47.6 24.3 1,260.4 1,345.6 506.5 706.7 616.3 315.6 F- 29 (Rs. in millions) For the Year Ended March 31 2000 2001 2002 2003 2004 Half year ended 30.09.2004 Interest Rate Swap MTM 0.0 0.0 0.0 0.3 (2.4) 216.8 Establishment Expenses 9.3 10.7 14.5 18.9 18.4 7.7 Administrative & Other Expenses 31.5 23.9 48.5 49.4 48.9 23.1 Share Issue Expenses written off 0.0 5.4 5.4 16.2 0.0 0.0 Provision against overdue call lending 0.0 50.0 0.0 0.0 0.0 0.0 Depreciation 0.0 0.0 0.0 0.0 0.0 0.0 - On Fixed Assets 3.9 3.9 6.3 9.4 9.4 4.7 0.0 0.0 0.0 0.0 0.0 0.0 TOTAL EXPENSES 1,305.1 1,439.5 581.2 800.9 690.6 567.9 Profit before Taxation 1,058.4 807.1 1,762.8 1,500.1 1,686.5 -871 Less: Provision for Taxation (including Deferred tax adjustment) 400.8 336.3 636.7 575.0 616.9 0.3 Profit after Taxation 657.6 470.8 1,126.1 925.1 1,069.6 -871.3 Balance Brought forward from last year 6.7 160.8 232.6 682.9 696.8 0 Amount Available for Appropriations 664.3 631.6 1,358.7 1,608.0 1,766.4 0 APPROPRIATIONS: General Reserve 250.0 36.0 112.6 92.6 107.0 0 Statutory Reserve Fund 131.6 95.0 225.2 185.1 214.0 0 Market Fluctuation Reserve 0.0 0.0 0.0 270.0 360.0 0 Interim Dividend 109.7 0.0 135.0 135.0 162.0 0 Proposed Dividend 0.0 243.0 189.0 202.5 175.5 0 Income Tax on Interim Dividend 12.2 0.0 13.8 0.0 20.7 0 Income Tax on Proposed Dividend 0.0 24.8 0.0 26.0 22.5 0 503.5 398.8 675.6 911.2 1,061.7 0 Balance Carried to Balance Sheet 160.8 232.8 683.1 696.8 704.7 0 F- 30 SUMMARIZED STATEMENT OF ASSETS AND LIABILITIES (Rs. in millions) AS AT March 31 2000 2001 2002 2003 2004 As at 30.09.2004 SOURCES OF FUNDS 1. Shareholders Funds: - Share Capital 1000.0 1350.1 1350.1 1350.1 1350.1 1350.1 - Reserves & Surplus 1105.1 2008.0 2812.8 3374.4 4063.2 3192.0 2105.1 3358.1 4162.9 4724.5 5413.3 4542.1 2. Loan Funds: - Secured Loans 8900.0 6310.0 3231.1 0.0 0.0 4538.3 - Unsecured Loans 5536.8 439.5 405.5 6986.6 9795.8 8124.9 14436.8 6749.5 3636.6 6986.6 9795.8 12663.2 TOTAL 16541.9 10107.6 7799.5 11711.1 15209.1 17205.3 APPLICATION OF FUNDS 1. Fixed Assets: Gross Block 45.7 56.8 83.9 101.4 113.2 114.9 Less: Depreciation 10.4 14.2 20.0 28.6 37.3 41.9 35.3 42.6 63.9 72.8 75.9 73.0 Capital Work in Progress 0.0 0.0 1.0 0.0 0.0 0.0 35.3 42.6 64.9 72.8 75.9 73.0 2. Investments 0.0 0.0 53.6 53.6 53.6 53.6 3. Current Assets, Loans and Advances: A. Current Assets (a) Stock in Trade 16006.5 9287.9 7001.7 11342.3 14983.6 17058.4 (b) Accrued Income 394.6 238.0 224.7 326.8 283.6 53.4 (c) Cash and Bank Balances 5.2 54.2 13.2 29.4 10.4 8.4 16406.3 9580.1 7239.6 11698.5 15277.6 15277.6 B. Loans & Advances 724.1 1496 1993 1687.5 1903.3 1382.9 Total Current Assets 17130.4 11076.1 9232.6 13386 17180.9 18503.1 Less: Current Liabilities & Provisions: - Liabilities 32.6 24.5 18.4 40.5 71.8 39.0 - Provisions 591.2 1008.2 1564.8 1775.3 2042.6 1398.4 623.8 1032.7 1583.2 1815.8 2114.4 1437.4 Net Current Assets 16506.6 10043.4 7649.4 11570.2 15066.5 17065.7 Deferred Tax 0.0 0.0 15.4 14.5 13.1 13.0 Misc. Expenditure 0.0 21.6 16.2 0.0 0.0 0.0 TOTAL 16541.9 10107.6 7799.5 11711.1 15209.1 17205.3 F- 31 SIGNIFICANT CHANGES IN ACCOUNTING POLICIES/MATERIAL NOTES TO ACCOUNTS QUALIFICATIONS IN AUDITORS’ REPORT DURING THE FIVE AND HALF YEAR PERIOD ENDING 30TH SEPTEMBER 2004. CHANGES IN ACCOUNTING POLICIES 1. During the Financial Year 2000-01, the company had changed its accounting policy for valuation of stock-in-trade. Accordingly, the company has adopted the rates provided by FIMMDA/PDAI for determining the market value of securities. Prior to the change, the market value of securities was determined on the basis of market rates as available at NSE/RBI SGL trades. Wherever the market rates were not available, the securities were valued at book rate. The change in accounting policy of valuation of stock-in-trade as mentioned above has resulted in reduced depreciation of Rs.23.010 million. Accordingly, profits of the Financial Year 2000-01 are higher to the said extent. 2. During the Financial Year 2001-02, the company had implemented a revised accounting policy on accounting for taxes on income in order to comply with the requirements of Accounting Standard 22 on “Accounting for taxes on income”. Accordingly, the accumulated deferred tax as on 01st April 2001 had been adjusted against the General Reserve and the adjustment for the Financial year 2001-02 amounting to Rs.1.292 million has been made out of the profits for the said financial year. The profits for the financial year 2001-02 have been reduced by the same amount. 3. During the Financial Year 2002-03, the company had revised its accounting policy with respect to amortisation of share issue expenses. Share issue expenses as per the revised accounting policy would be amortised in the year in which it is incurred as against the earlier policy of amortising the same over a period of five years. Accordingly, an additional write- off of Rs.10.786 million has been made during the Financial Year 2002-03. Consequently, the profit for the Financial Year 2002-03 is understated to the said extent. 4. During the Financial Year 2003-04, in terms with the requirements of AS-26 on accounting for Intangible Assets, software acquired in the earlier years which was included as part of Computers has been classified under the head “Intangible Assets”. During the said financial year, the company has amortized a sum of Rs.1.027 million. The amortisation rate adopted is consistent with the practice followed in the earlier years, hence, it has no impact on the current year’s profitability. 5. During the Financial Year 2003-04, in conformity with the Guidelines formulated by RBI (applicable from 01st April 2003), the company has revised the method for valuation of securities under repo and reverse repo contracts outstanding as at the year-end. Under the new method, provision for contracts wherein second leg of repo transaction is outstanding, is lesser by Rs.0.551 million as compared to the earlier practice. Accordingly, the profits of the Financial Year 2003-04 are higher to the said extent. 6. During the Financial Year 2003-04, the company had initiated transactions in Interest Rate Swaps (IRS). To account for the same, the company has adopted the following accounting practice: Assets and Liabilities in respect of notional principal amount of IRS is nullified. The related interest is recognized on accrual basis. Any other charges relating to Interest Rate Swaps are charged to Profit and Loss Account. Trading Interest Rate Swaps outstanding at balance sheet date are marked to market and the resultant loss, if any, is recorded in Profit and Loss Account. MATERIAL NOTES TO ACCOUNTS 1. An amount of Rs.100 million was lent in Call Money to Madhavpura Mercantile Cooperative Bank Limited, which was overdue as on 31st March 2001. Therefore, a provision of Rs.50 million had been made against the same during the Financial year 2000-01. In the Financial Year 2001-02, a reconstruction sheme was formulated under the aegis of Government of India and the lending inclusive of interest upto date of implementation of scheme has been converted into term deposit of Rs.103.658 million carrying interest at the rate of 7.50% (payable semi-annually) and principal repayable in 5 equal installments starting from the 5th year. However, as a matter of prudence, the existing provision has not been reversed till date. 2. The company had purchased and obtained possession of three Residential flats in Chennai for Rs.5.196 million during the Financial Year 2002-03 from Tamil Nadu Housing Board (TNHB). The TNHB has not decided the amount of stamp duty leviable on the registration of said property. In the absence of any decision in this regard by the Housing Board, the F- 32 amount of the stamp duty remains unascertained and the flats are yet to be registered in the company’s name. 3. During the Financial Year 2002-03, the company has voluntarily decided to build a special reserve over a period of time designated as “Market Fluctuation Reserve” equivalent to a maximum of the equity capital to meet market fluctuations. Accordingly, a sum of Rs.270 million has been appropriated towards market fluctuation reserve out of the net profits of the Financial Year 2002-03 and Rs.360 million has been appropriated out of the net profits of the Financial Year 2003-04, thereby allocating a total of Rs.630 million. QUALIFICATIONS IN AUDITORS’ REPORT There have been no statements in the nature of qualifications in the Auditors’ Reports during the five and half year period ending 30th September 2004. ACCOUNTING POLICIES AND NOTES FORMING PART OF THE ACCOUNTS AS ON 30.09.2004 A. SIGNIFICANT ACCOUNTING POLICIES 1. Method of Accounting The company follows accrual system of accounting and the financial statements are prepared on historical cost basis, in accordance with generally accepted accounting principles and Reserve Bank of India guidelines as applicable to the Primary Dealers. 2. Sales / Purchases of Treasury Bills and Government Dated Securities, as disclosed in Profit & Loss Account includes repo transactions. 3. Revenue Recognition i). The difference between the acquisition cost and maturity value of Certificates of Deposit, Commercial Papers, Bills Re-discounted, Treasury Bills and Zero Coupon Bonds is apportioned on time basis. The above is recognised as accrued income and included in the carrying cost of the securities. ii). Interest accrued on Government Dated Securities and Corporate Bonds and Debentures is recognised at its coupon rate and that of floating rate bonds is recognised on the yield of instruments to which these are linked. iii). Purchase and sale price of fixed income securities is bifurcated into cost and accrued interest paid or realised. Accrued interest paid on purchase & received on sale is netted and reckoned as expense/income. iv). Profit/loss on sale of securities is accounted on weighted average cost method and is recognised on settlement date. Profit on sale of securities is netted with loss on sale of securities. v). Brokerage and front-end fee received on subscription of securities is deducted in arriving at the cost of relevant securities. Underwriting fee earned in respect of devolvments in respect of underwriting commitments is proportionately reduced from the cost of securities devolving and the remaining amount is directly recognised as income. 4. Expenses Recognition The brokerage paid in connection with acquisition of securities is added to the cost of acquisition and on sale of securities it is charged to profit & loss account. 5. Valuation of Stock-in-Trade i). All securities in which the Company deals are regarded as Current Assets (Stock-in-Trade). ii). Each type of Central Government, State Government securities and Corporate Bonds and Debentures is regarded as a separate category. Each category of securities is valued scripwise and depreciation or appreciation is aggregated under each category. Net depreciation, if any, for each category of securities is provided for and charged to profit and loss account. Net appreciation, if any, is ignored. The depreciation in one category of securities is not set off against appreciation in another category. The market value of Central Government, State Government securities and Corporate Bonds & Debentures is determined as per the rates provided by FIMMDA/PDAI. iii). Certificates of Deposit, Commercial Papers, Bills Re-discounted, Treasury Bills and Zero Coupon Bonds held on the balance sheet date are valued at carrying cost. F- 33 6. Accounting for repo Transactions Securities sold under repo transactions are excluded from stock-in-trade and the securities purchased under reverse repo are included in the stock-in-trade. The securities purchased under reverse repo is valued in accordance with the valuation norms applicable for the respective category of security. In case of securities sold under repo, the notional loss, if any, arising out of the difference between the transaction price and book value in the first leg of the repo transaction is provided for, notional gains if any are ignored . 7. Interest Rate Swaps (IRS) Assets and Liabilities in respect of notional principal amount of IRS is nullified. The related interest is recognised on accrual basis. Any other charges relating to Interest Rate Swaps are charged to Profit and Loss Account. Trading Interest rate swaps outstanding at balance sheet date are marked to market and the resultant loss, if any, is recorded in Profit and Loss Account. 8. Investment Long Term Investment in debt is valued at carrying cost. However, provision for diminution is made, when there is a decline other than temporary in the value of long term investment. 9. Deferred Tax Deferred tax is recognized in accordance with the provisions of Accounting Standard 22 issued by Institute of Chartered Accountants of India on “Accounting for Taxes on Income”. 10. Depreciation Depreciation on fixed assets is charged on written down value method in accordance with the rates specified in Schedule XIV to the Companies Act, 1956. Intangible Assets comprises of software acquired by the company to facilitate its operations these are depreciated @40% on WDV basis. 11. Preliminary Expenses Preliminary expenses are written off in the year in which these are incurred. 12. Share Issue Expenses Share issue expenditure is charged to Profit and Loss account in the year it is incurred. 13. Tax on Dividend Corporate Dividend Tax payable on dividend declared in terms of Section 115-O of the Income Tax Act, 1961, is accounted for in the year to which the dividend relates. 14. Retirement Benefits – Provident Fund, Gratuity & Leave Encashment (i) Gratuity contribution made under the Employee Group Gratuity cum life insurance scheme of LIC is charged to revenue. (ii) Leave Encashment is accounted for on acturial valuation carried at year- end. (iii) Contribution to recognised provident fund is charged to revenue. F- 34 B. NOTES TO ACCOUNTS 1. In terms of Accounting Policy No 5(ii) the company has adopted rates provided by FIMMDA/PDAI for determining market value of securities. Accordingly, net depreciation in the value of securities as on 30th September 2004 amounts to Rs. 1.457 million. The same stands fully provided for in the valuation of the closing stock. 2. Repo transactions outstanding are as under: (Rs. in million) As at 30 September 2004 Face Value Book Value Purchases (Rev. Repo) NIL NIL Sales(Repo) 2494.00 2489.116 In terms with RBI guidelines for uniform accounting for Repo Transactions, there has been no loss on outstanding repo transactions as on 30th September 2004. 3. Managerial Remuneration for the Managing Director For the half-year 30th Sep 2004 (Rs. in million) Salaries and allowances 0.192 Company’s contribution to Provident Fund 0.013 Other perquisites 0.023 Computation of Net Profits under Section 349 of the Companies Act, 1956 has not been made as commission by way of percentage of profits is not payable to the Managing Director. 4. In the interest tax case involving an amount of Rs.3.177 million, the appellate authorities had adjudicated in favour of the company and a refund of Rs.2.867 million was received along-with interest of Rs.1.886 million during FY 2003-04. The company had preferred appeal for the remaining amount which is yet to come up for hearing. 5. The interest on the term deposit of Rs. 103.658 million with Madhavpura Mercantile Cooperative Bank has been serviced regularly. However, as a matter of abundant caution being guided by the accounting concept of prudence, the management has decided not to reverse the existing provision of Rs. 50 million made in earlier year. 6. Reportable segments in respect of business operations of the company have been identified on the basis of varied risk and return profile attached to each segment. During the half-year ended 30th September 2004, the company had identified “Derivatives” as an additional segment for reporting purposes in terms with Accounting Standard – 17 of the Institute of Chartered Accountants of India on Segment Reporting. F- 35 The Segment information is as under: Rs in million Half year ended 30th September 2004 T-Bills / Corporate Government Derivatives Others Total Commercial Bonds Securities Paper/ & Certificate Debentures of Deposit Segment Revenue/(Loss) 203.264 (8.468) (508.803) (208.812) 10.900 (511.919) Total Allocable Expenses 138.532 15.234 170.502 8.008 0.014 332.290 Segment Results 64.732 (23.702) (679.305) (216.820) 10.886 (844.209) Less : Unallocable Expenses 26.763 Profit/(Loss) Before Tax (870.972) Segment Assets * 12598.362 178.782 4335.215 (15.837) 104.446 17200.968 Segment Liabilities 9263.127 131.452 3187.529 0.004 76.796 12658.908 Note: Depreciation of Rs. 1.457 million on Government Securities as on 30.09.2004 has been provided for. * Segment assets under Others comprise of investments and interest accruals thereupon 7. Related Party Transactions i. As per Para 9 of the Accounting Standard 18 on Related Party Disclosures, the company being a state controlled enterprise is not required to make disclosures of related party relationships with other state controlled enterprises and transactions with such enterprises. Other information as per the Standard is as under: a. The overall supervision and control of company vests with Board of Directors. The Managing Director of the company is on deputation from Punjab National Bank and is working full time with the company. Details of managerial remuneration is disclosed vide Note No. 3 above in the Notes to Accounts. b. Out of a total of 9 directors on the Board of the company as at 30th September 2004, 5 are independent directors. Only the independent directors are paid sitting fees for the board / committee meetings at the rate of Rs 3000/ - per meeting. During the current reporting period, the company has paid a sum of Rs 0.118 million towards sitting fee. 8. Earnings per share (EPS) Computation of EPS is as under: Half year ended 30.09.2004 Number of shares outstanding (of Rs 10/- each) 135007600 Profit/(Loss) after Tax ( Rs in million) (871.266) Earning per share (Basic) (6.45) There has been no change in the share capital during the reporting period. There are no potential equity shares outstanding. Hence there is no dilution of the Basic EPS. F- 36 9. Deferred Tax Calculation of deferred tax as per Accounting Standard 22 of The Institute of Chartered Accountants of India is as under: Rs in million September 30, 2004 Deferred Tax Asset Provision against Investments 18.296 Other Provisions 0.145 Preliminary Expenses 0.441 Total Deferred Tax Asset 18.882 Deferred Tax Liability Fixed Assets 5.844 Total Deferred Tax Liability 5.844 Net Deferred Tax Asset 13.038 An adjustment for the current half-year amounting to Rs. 0.094 million has been charged to P&L Account. 10. Disclosure on Interest Rate Swaps Amt Rs in million Notional Principal (Trading Swaps) 84750.000 Fair Value 84592.334 Associated Credit Risk 423.750 Default Risk (as per Credit Exposure Method) 1021.358 Market risk In the event of 100 basis point adverse movement in interest rates there will be a negative impact of Rs. 132.200 million on the swap book. Collateral No Collateral is insisted upon from counterparty 11. During the earlier year, in conformity with the Guidelines formulated by RBI (applicable from 1st April, 2003), the company has revised the method for valuation of securities under repo and reverse repo contracts outstanding as at the year-end. The same method has been continued during the present half-year also. 12. In the previous year ended 31.03.2004, in terms with the requirements of AS-26 on accounting for Intangible Assets, software acquired in the earlier years which was included as part of Computers had been classified under the head “Intangible Assets”. The amortisation rate adopted was consistent with the practice followed in the earlier years, hence, it had no impact on the profitability. During the current half-year also, the same amortisation rate adopted earlier has been followed. 13. In the absence of any government notification the company has not provided for cess envisaged in terms with the Section 441A (1) of the Companies Act, 1956. 14. The company had purchased and obtained possession of three Residential flats in Chennai for Rs. 5.196 million during 2002-03 from Tamil Nadu Housing Board (TNHB). The TNHB has not decided the amount of stamp duty leviable on the registration of said property. In the absence of any decision in this regard by the Housing Board, the amount of the stamp duty remains unascertained and the flats are yet to be registered in the company’s name. 15. Contingent liability: - Claims made against not acknowledged as debts Rs. 0.377 million F- 37 ANNEXURE D GROVER, LALLA & MEHTA CHARTERED ACCOUNTANTS The Managing Director, PNB Housing Finance Ltd. 9th Floor, Antriksh Bhawan, 22 Kasturba Gandhi Marg, New Delhi – 110 001 Dear Sirs, In terms of our appointment for the purpose of certification of statement of accounts to be incorporated in the offer document proposed to be issued by Punjab National Bank in connection with the follow on offer of Equity Shares, we state as follows:- 1. We have examined the audited financial statements of PNB Housing Finance Ltd. for the five financial years ending 31.03.2004, being the last date up to which the accounts have been made and audited by the statutory auditors of the company of those respective years and Financial Statements for the half year ending 30th September, 2004 which have been compiled from the unaudited half yearly returns received from the twenty eight branches and books maintained at Corporate Office at New Delhi. We clarify that financial statements are the responsibility of the Company’s management and these financial statements for the six months ending 30th September 2004 have not been subjected by us to a complete audit, in accordance with the generally accepted auditing practices necessary for expressing an opinion whether the statements show a true and fair view. 2. The aforesaid financial statements read with significant Notes on Accounts have been prepared in accordance with the prudential norms issued by the National Housing Bank from time to time and subject to limitation of disclosure required therein. We have however conducted a limited review for the six months ending 30th September 2004, to obtain moderate assurance as to whether the financial statements are free of material mis- statements. A review is limited primarily to inquiries of company personnel and procedures applied to financial data and thus provides less assurance than an audit. We have not performed an audit and accordingly, we do not express an opinion on the same. 3. Based on the limited review for the six months ending 30th September, 2004, conducted by us as aforesaid, nothing has come to our notice that causes us believe that the accompanying statements do not give a true and fair view in accordance with the accounting standards, other recognized accounting policies and practices and relevant statutory requirements. For Grover, Lalla & Mehta Chartered Accountants Sd/ (A.K.Grover) Partner Place: New Delhi Date: 13-10-2004 F- 38 PNB HOUSING FINANCE LIMITED PART – 1: COMPARATIVE PROFIT AND LOSS ACCOUNT (Rs in millions) YEAR ENDED ON 31ST MARCH PERIOD ENDED ON INCOME 2000 2001 2002 2003 2004 30.06.04 30.09.04 Interest on Housing Loans 334.7 412.4 565.1 691.5 773.1 190.6 377.8 Interest on Investments 18.9 68.0 52.2 53.6 63.2 13.3 26.3 Interest on Bank Deposits 30.3 3.4 0.9 1.8 0.0 0.1 2.5 Int.on Loans Against Company Deposits 1.3 1.3 1.2 1.3 1.8 0.2 0.7 Fee and Other Charges 16.4 28.0 48.7 49.5 49.2 11.1 22.4 Other Income 1.7 1.9 0.3 1.2 6.0 0.4 0.7 Profit on sale of Assets 0.0 0.0 -0.1 0.2 0.0 0.0 0.0 Profit on sale of Investments 4.5 2.1 0.0 3.8 10.6 0.0 0.0 Dividend 6.8 7.0 0.9 0.8 0.5 0.0 0.0 Recovery of Int.derecognised 0.0 0.0 0.0 0.0 0.0 0.0 0.0 in previous year Recovery of Debt Written off 92.6 0.0 0.0 0.0 0.0 0.0 0.0 TOTAL INCOME 507.2 524.1 669.2 803.7 904.4 215.7 430.4 EXPENDITURE Interest on T.loans/overdraft 68.1 64.1 110.6 87.1 126.0 31.6 66.1 Interest on Refinance from NHB 39.7 90.3 156.4 204.3 191.4 38.6 84.9 Interest on Bonds 0.0 0.0 22.4 90.7 102.5 24.0 47.6 Interest on Deposits 189.9 175.9 165.5 168.9 167.2 39.9 75.7 Bank and Commitment charges 5.2 4.6 8.4 4.5 6.9 0.6 1.5 Interest Tax 7.4 0.0 0.0 0.0 0.0 0.0 0.0 Staff Expenses 14.1 17.8 19.9 24.8 31.0 8.0 16.7 Rent, Rates & Taxes 3.3 3.7 5.6 8.1 8.5 1.8 4.4 Repairs and Maintenance 1.5 1.5 4.1 4.3 2.6 0.5 1.4 Electricity & Water Charges 1.1 1.2 2.0 2.6 2.9 0.5 1.7 Insurance 0.1 0.1 0.2 0.2 0.3 0.1 0.3 Travelling and Conveyance 1.0 1.4 2.1 2.8 3.8 0.9 2.4 Printing & Stationery 1.0 1.1 1.5 1.3 1.7 0.3 0.9 Postage & Telephone 0.9 1.2 1.8 2.2 3.3 0.6 1.9 F- 39 (Rs in millions) YEAR ENDED ON 31ST MARCH PERIOD ENDED ON 2000 2001 2002 2003 2004 30.06.04 30.09.04 Advertisements 1.7 4.6 11.0 7.2 9.0 0.8 4.0 Loss on sale of Assets 0.0 0.0 0.0 0.0 0.0 0.0 0.0 General Office Expenses 1.1 1.5 1.9 3.3 3.5 0.9 1.7 Auditors Remuneration 0.1 0.2 0.3 0.3 0.5 0.0 0.0 Professional and Legal Charges & Other Certification charges 0.7 0.8 2.0 3.8 3.7 1.3 3.1 Depreciation on Fixed Assets 2.1 3.1 4.9 5.9 6.9 2.1 4.2 Depreciation on Investments 0.0 9.0 0.0 0.0 0.0 0.0 0.0 Misc. Expenses 0.8 0.7 0.6 0.9 0.6 0.5 0.3 Interest Derecognised 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Provision for NPA 9.9 6.3 10.7 8.8 20.1 5.0 14.0 Provision for Contingencies 0.1 0.0 0.0 0.0 0.0 0.0 0.0 Bad Debts Written off 73.4 8.7 14.2 8.0 0.0 0.0 0.0 Preliminary Exp.written off 0.0 0.0 0.0 0.0 0.0 0.0 0.0 TOTAL EXPENDITURE 423.2 397.8 546.1 640.0 692.4 158.0 332.8 Profit (Loss)Before Tax 84.0 126.3 123.1 163.7 212.0 57.7 97.6 Prior Period Adjustments -1.0 -0.6 0.4 0.2 -0.1 0.0 0.0 Provision for Income Tax of 0.0 0.0 0.8 -4.9 -2.4 0.0 0.0 earlier years written back Provision for Income Tax 26.7 27.0 28.0 45.3 60.3 15.2 27.7 Deferred Tax 0.0 0.0 4.5 -5.5 -5.7 0.0 -7.6 Profit (Loss)after Tax/ Profit available for Apportionment 56.3 98.7 91.8 119.2 154.9 42.5 77.5 Surplus Brought forward from Prev. Year 13.3 2.3 6.0 2.5 10.3 18.4 18.4 Transfer to Special Reserves u/s 36(1)(viii) of Income Tax Act, 1961 29.0 28.2 42.3 59.2 74.6 0.0 0.0 Transfer to General Reserves 5.0 30.0 20.0 15.0 35.0 0.0 0.0 Dividend on Equity shares 30.0 30.0 33.0 33.0 33.0 0.0 0.0 Dividend Distribution Tax 3.3 6.8 0.0 4.2 4.2 0.0 0.0 Surplus Carried to Balance Sheet 2.3 6.0 2.5 10.3 18.4 60.9 95.9 F- 40 STATEMENT OF ASSETS AND LIABILITIES PART-II : COMPARATIVE BALANCE SHEET (Rs. millions) AS ON 31ST MARCH AS ON AS ON 2000 2001 2002 2003 2004 30.06.04 30.09.04 LIABILITIES Share Capital 300.0 300.0 300.0 300.0 300.0 300.0 300.0 Special Reserves 167.7 195.9 239.8 299.0 373.7 373.7 373.7 General Reserves 70.0 100.0 133.8 148.8 183.7 183.7 183.7 Profit & Loss Account 2.3 6.0 2.5 10.3 18.4 60.9 95.9 Term Loan from NHB 348.7 1131.9 1558.1 1827.1 1904.2 2889.4 1794.0 Term Loan from PNB 585.0 555.0 1365.0 700.0 1340.6 826.2 1356.4 Term Loan from Other Banks 0.0 0.0 0.0 653.7 1057.4 1018.6 1479.7 Commercial paper 0.0 0.0 0.0 0.0 0.0 247.0 246.8 Secured Redeemable Bonds 0.0 0.0 500.0 1250.0 1250.0 1250.0 1250.0 Interest Accrued and Due on Deposits 7.5 6.5 8.0 9.8 10.6 1.8 9.7 Short Terms Loans from PNB/NHB 0.0 0.0 0.0 0.0 0.0 0.0 120.0 Home Loan Account 0.3 0.3 0.2 0.0 0.0 0.0 0.0 Non-Cumulative Deposit 176.1 158.2 190.3 259.1 334.3 469.9 310.2 Cumulative Deposit 1095.2 1005.5 1197.6 1266.7 1205.4 1035.0 1143.4 Current Liabilities 333.6 357.6 281.9 319.7 387.4 392.3 285.0 Provisions 16.6 23.1 65.2 78.8 99.4 104.4 76.2 TOTAL LIABILITIES 3103.0 3840.0 5842.4 7123.0 8465.1 9152.9 9024.7 ASSETS Fixed Assets 7.5 23.5 30.3 29.4 31.9 30.6 29.5 Investments 595.0 502.6 431.2 431.2 546.3 423.7 423.7 Housing Loans 2005.3 2912.6 4919.0 6261.9 7545.5 7860.6 8210.1 Loans and Advances 318.9 340.7 335.1 199.6 243.7 313.6 303.1 Cash In Hand 0.5 0.6 1.3 2.5 3.6 1.2 1.6 Cash at Bank 87.0 29.9 114.1 143.5 32.4 -10.5 -37.6 Fixed Deposit 86.1 30.0 0.0 0.0 0.0 460.0 0.0 Other Current Assets 2.7 0.1 10.9 54.9 61.7 73.7 94.3 Miscellaneous Expenses to the extent not written off 0.0 0.0 0.5 0.0 0.0 0.0 0.0 TOTAL ASSETS 3103.0 3840.0 5842.4 7123.0 8465.1 9152.9 9024.7 F- 41 (Rs. millions) AS ON 31ST MARCH AS ON AS ON 2000 2001 2002 2003 2004 30.06.04 30.09.04 Contingent Liability not provided for : 1. For Contracts unexecuted o/s 5.4 0.1 1.5 1.3 0.2 0.2 0.1 2. For Others 0.1 0.1 0.00 0.00 0.00 0.00 0.00 3. Income Tax matter under dispute * 42.00 46.9 62.8 43.3 23.3 23.3 26.2 4. Interest Tax matter under dispute * 3.8 5.4 6.1 4.1 4.1 4.1 4.1 * The company has paid advance taxes and as per the opinion of tax consultants, the company has a good case and the disallowances made by the Tax Authorities are arbitrary and appropriate reliefs will be available at appellate level. Hence no provision was considered necessary. For PNB HOUSING FINANCE LTD. (SANJAY JAIN) EVP (F) & CO. SECRETARY F- 42 SIGNIFICANT ACCOUNTING POLICIES 1. GENERAL METHOD AND SYSTEM OF ACCOUNTING The accounts of the company are prepared under the Historical Cost convention on accrual method of accounting on the principle of going concern and in accordance with the Companies Act, 1956, the applicable accounting standards issued by The Institute of Chartered Accountants of India, and The Housing Finance Companies (NHB) Directions, 2001 issued by National Housing Bank. 2. (a) FEE AND OTHER CHARGES Income from fee and other charges viz. Administrative Fee, Processing Fee, Penal Interest on Overdues /Additional Interest on Defaults, Pre-payment charges etc. is recognised on receipt basis. (b) DIVIDEND INCOME Dividend is accounted for in the year in which the same is received. (c) OTHER INCOME Interest on tax refunds and other incomes are accounted for on receipt basis. (d) INTEREST ON HOUSING LOANS In housing loans, the repayment is received by way of Equated Monthly Instalments (EMIs) comprising principal and interest. Interest is calculated on the outstanding balance at the beginning of the year/ month. EMIs generally commence once the entire loan is disbursed. Pending commencement of EMIs, Pre-EMI interest is charged every month. 3. UNCLAIMED DEPOSITS Deposits, which have become due but have not been presented for payment or renewal are transferred to unclaimed deposits. Interest for the period from last maturity date till the date of renewal of unclaimed deposits is accounted for during the year of its renewal. 4. INVESTMENTS Investments are capitalised at cost inclusive of brokerage and stamp charges excluding interest/dividend accruing till the date of purchase. The difference between the carrying amount and disposal proceeds of investments, net of expenses, is recognised in the Profit & Loss Account. Investments are classified as long term investments and current investments and are valued in accordance with guidelines of National Housing Bank and Accounting Standard on Accounting for Investments (AS-13), issued by The Institute of Chartered Accountants of India. Long term investments are valued at cost. However, when there is a decline, other than temporary, in the value of long term investment, the carrying amount is reduced to recognise the decline. Current investments have been valued at lower of cost or market value determined on individual investment basis. 5. DERIVATIVE TRANSACTIONS (i) The derivative transactions entered for hedging interest bearing liabilities are accounted for on accrual basis. (ii) Gains or losses on swaps are accounted for on the date of unwinding and are adjusted in the Interest Expenditure account. 6. FIXED ASSETS (i) Fixed Assets used in the business are capitalised at cost inclusive of all related expenses. (ii) As per the National Housing Bank Directions 2001, the land and buildings, including Assets / Properties acquired from NPA Advances in settlement of Housing Loans, are held at cost as “ Other Current Assets” till their disposal. These assets are required to be disposed off within 3 years from the date of acquisition unless and until extended by the NHB. All the expenses incurred on the upkeep of the property including safeguarding , insurance, rates & taxes etc are charged to Profit & Loss Account in the year of incurrence. 7. DEPRECIATION (i) Depreciation on Fixed Assets is provided on the Written Down Value Method at the rates prescribed in Schedule XIV of the Companies Act, 1956 on prorata basis. Wherever the depreciation rates are not prescribed, the rates F- 43 prescribed by Income Tax Act & Rules are used. (ii) Assets costing upto Rs. 5000/- are being depreciated fully in the year of acquisition. 8. SHARE/ BOND ISSUE EXPENDITURE Expenditure incurred on issue of shares/bonds is charged to Profit & Loss Account in the year it is incurred. 9. TAXES ON INCOME Current tax is determined as the amount of tax payable in respect of the taxable income for the period. Deferred tax is recognised in accordance with the provisions of Accounting Standard on Accounting for taxes on income (AS-22). 10. RETIREMENT BENEFITS AND LEAVE ENCASHMENT (i) The company has taken LIC Policy to cover the accumulated gratuity liability till 30.9.2004 of its employees. Premium on this policy has been accounted for on accrual basis in line with the Accounting Standard on Accounting for Retirement Benefits (AS-15), issued by the Institute of Chartered Accountants of India. (ii) Provision for leave encashment is made on the basis of actuarial valuation with respect to privilege leaves which are encashable. (iii) Retirement benefits of employees on deputation from Punjab National Bank are borne by PNB and hence no provision is considered necessary by the company. 11. INSTALMENTS - ACCOUNTING OF LOANS (i) Loans to the extent, the instalments have not become due as at the year end are being shown under “Housing Loans.” (ii) Loans to the extent the instalments have become due on or before 30th September, 2004 are being shown under “Loans and Advances”. 12. PRUDENTIAL NORMS Income recognition and Provision for Non Performing Assets have been made on the basis of prudential norms laid down by National Housing Bank (NHB). 13. TRANSACTION INVOLVING FOREIGN EXCHANGE (i) Foreign currency monetary liabilities are translated at the rate which reflects the liability of the company in Indian Rupee which is likely to be repaid at the balance sheet date. (ii) Income and expenditure items are accounted for at the exchange rate prevailing on the date of transaction. (iii) Generally Exchange differences arising on Foreign Currency transactions are recognised as income or expense as the case may be in the period in which they arise. However, in case of forward exchange contracts, the Exchange difference between the forward rate and the exchange rate at the date of transaction is recognised as an income or expense over the life of the forward contract in line with Accounting Standard on Accounting for the Effects of Changes in Foreign Exchange Rates (AS-11). F- 44 SIGNIFICANT CHANGES IN ACCOUNTING POLICIES This is to certify based upon our examination and review of audited accounts of PNB Housing Finance Ltd., New Delhi from financial years 1999-2000 to 2003-2004, that there is only one change in the significant accounting policies, which is given below : Share/ Bond Issue Expenditure Accounting Policy on Share/ Bond Issue Expenses- as deferred Revenue Expenses was changed in the year 2002-03. Now such expenses are written off in the year of incurrence. Material Adjustments resulting from Statutory Auditor’s Report for previous years are as under :- (Rs. in millions) Year ended 31st March 2000 2001 2002 2003 2004 Net Profit for the year 56.3 98.7 91.8 119.2 154.9 Less : Adjustments 0.00 0.00 0.00 0.00 0.00 Adjusted Profit 56.3 98.7 91.8 119.2 154.9 NOTES TO ACCOUNTS With regards to notes on accounts of PNB Housing Finance Ltd. New Delhi for its audited accounts for the years 1999-2000 to 2003-2004, we observe that most of the notes are explanatory in nature and have been included in the relevant schedule to the respective year’s audited accounts due to disclosure requirements of Companies Act 1956, Accounting Standards and NHB Guidelines, relevant regulations and management’s disclosure to the reader of Accounts. Year 1999-2000 Note 11: Contingent liabilities not provided for Rs in millions Particulars As at As at March 31, 2000 March 31, 1999 - Estimated amount of contracts remaining unexecuted (net of advance) 5.4 3.2 - For Others 0.1 0.2 - Income tax matters under dispute 42.0 29.0 - Interest tax matters under dispute 3.8 3.0 (the company has paid advance taxes and as per the opinion of tax consultants, the company has a good case and the disallowances made by the Tax Authorities are arbitrary and appropriate relief will be available at appellate level. Hence no provision was considered necessary.) Year 2000-01 Note 1 : The age-wise analysis of loans in respect of which the instalments (including interest) have become due are as under:- Rs in millions Particulars For the year Ended For the year Ended March 31, 2001 March 31, 2000 Outstanding for over six months 235.1 200.8 Outstanding for six months or less 61.1 73.3 296.2 274.1 F- 45 Note 3 : Provision for non-performing assets has been made in accordance with guidelines on prudential norms issued by National Housing Bank (NHB). Classification of loans and provisions made for non-performing assets is given hereunder: (Rs. in millions) Loans Standard Sub Standard Doubt-ful Total Housing Loans (including instalments due from borrowers) Individuals 2757.7 21.0 0.9 2779.6 (Previous Year) (1766.5) (9.6) (0.6) (1776.7) Corporate Bodies 292.1 77.0 60.0 429.1 (Previous Year) (366.5) (141.0) (5.0) (512.5) Other Loans 9.9 0.00 0.00 9.9 (Previous Year) (9.3) (0.00) (0.00) (9.3) Total Loans 3059.7 98.0 60.9 3218.6 (Previous Year) (2142.3) (150.6) (5.6) (2298.5) Provisions (as per NHB guidelines) 0.00 9.8 12.7 22.5 (Previous Year) (0.00) (15.0) (1.1) (16.1) Total Provisions 0.00 9.8 12.7 22.5 (Previous Year) (0.00) (15.0) (1.1) (16.1) Note 4 : Interest on non-performing assets is recognized on realization basis as per the NHB Guidelines. Accordingly the total interest not recognized as at the Balance Sheet date is summarized as under:- Particulars Rs in millions Cumulative interest B/F from last Balance Sheet 83.7 Less: Recovered/ written back during the year 55.1 28.6 Add: Interest not recognised for the current year on Sub Standard Assets 17.8 - Doubtful Assets 7.5 Total interest not recognised as on 31.03.2001 53.9 Note 5 : The company has entered into “ One Time Settlement” (OTS) with Allahabad Development Authority wherein, interalia, U.P.Government were to issue following Bonds during the year w.e.f. 1-4-2000 (a) 10% Bonds of Rs.50 millions (Redeemable in 10 Equal Annual Instalments) and (b) 0% Bonds of Rs 62.5 millions (Redeemable in 6 Equal Annual Instalments). Despite vigorous follow up by the company, U.P.Govt. has not yet issued these bonds on account of procedural & Administrative delays. However, the interest on these bonds alongwith Annual Instalments of redemption, as per OTS terms, for both kinds of Bonds, have been received during the year. U.P.Govt. has assured that the Bonds for the balance amount due will be issued shortly and will be effective from 1-4-2001. Pending issuance of Bonds the remaining outstandings have been included in “Loans & Advances” in Schedule 7 as shown in the previous year. Note 8 : Contingent liabilities not provided for Rs in millions Particulars As at As at March 31, 2001 March 31, 2000 - Estimated amount of contracts remaining unexecuted (net of advance) 0.1 5.4 - For Others 0.1 0.1 - Income tax matters under dispute* 46.9 42.0 - Interest tax matters under dispute* 5.3 3.8 * As advised by Tax Consultant, no provision has been made. F- 46 Year 2001-02 Note 1 : The age-wise analysis of loans in respect of which the instalments (including interest) have become due are as under: - Rs in millions Particulars As at As at March 31, 2002 March 31, 2001 Outstanding for over six months 200.4 235.1 Outstanding for six months or less 80.3 61.1 280.7 296.2 Note 3 : Provision for non-performing assets has been made in accordance with guidelines on prudential norms issued by National Housing Bank (NHB). Classification of loans and provisions made for non-performing assets is given hereunder: (Rs. in millions) Loans Standard Sub Standard Doubt-ful Total Housing Loans (including instalments due from borrowers) Individuals 4832.8 76.4 5.5 4914.7 (Previous Year) (2757.7) (21.0) (0.9) (2779.6) Corporate Bodies 157.1 52.2 75.6 284.9 (Previous Year) (292.1) (77.0) (60.0) (429.1) Other Loans 13.4 0.00 0.00 13.4 (Previous Year) (9.9) (0.00) (0.00) (9.9) Total Loans 5003.3 128.6 81.1 5213.0 (Previous Year) (3059.7) (98.0) (60.9) (3218.6) Provisions (as per NHB guidelines) 0.00 12.9 18.3 31.2 (Previous Year) (0.00) (9.8) (12.7) (22.5) Total Provisions 0.00 12.9 18.3 31.2 (Previous Year) (0.00) (9.8) (12.7) (22.5) Note 4 : Interest on non-performing assets is recognized on realization basis as per the NHB Guidelines. Accordingly the total interest not recognized as at the Balance Sheet date is summarized as under:- Particulars Rs in millions Cumulative interest B/F from last Balance Sheet 53.9 Less: Recovered/ written back/ written off during the year 21.3 32.6 Add: Interest not recognised for the current year on - Sub Standard Assets 9.2 - Doubtful Assets 14.7 Total interest not recognised as on 31.03.2002 56.6 F- 47 Note 14 : Contingent liabilities not provided for Rs in millions Particulars As at As at March 31, 2002 March 31, 2001 - Estimated amount of contracts remaining unexecuted (net of advance) 1.5 0.1 - For Others 0 0.1 - Income tax matters under dispute* 62.8 46.9 - Interest tax matters under dispute* 6.1 5.3 * As advised by Tax Consultant, no provision has been made. Year 2002-03 Note 1 : The age-wise analysis of loans in respect of which the instalments (including interest) have become due are as under :- Rs in millions Particulars As at As at March 31, 2003 March 31, 2002 Outstanding for over six months 97.5 200.4 Outstanding for six months or less 57.9 80.3 155.4 280.7 Note 3 : Provision for non performing assets has been made in accordance with guidelines on prudential norms issued by National Housing Bank (NHB). Classification of loans and provisions made for non performing assets is given hereunder : (Rs. in millions) Loans Standard Sub Standard Doubtful Total Housing Loans (including instalments due from borrowers) Individuals 6169.1 163.4 12.4 6344.9 (Previous Year) (4832.8) (76.4) (5.5) (4914.7) Corporate Bodies 12.1 0.00 60.3 72.4 (Previous Year) (157.1) (52.2) (75.6) (284.9) Other Loans 18.5 0.00 0.00 18.5 (Previous Year) (13.4) (0.00) (0.00) (13.4) Total Loans 6199.7 163.4 72.7 6435.8 (Previous Year) (5003.3) (128.6) (81.1) (5213.0) Provisions (Housing Loan) 0.00 16.3 23.7 40.0 (Previous Year) (0.00) (12.9) (18.3) (31.2) Total Provisions 0.00 16.3 23.7 40.0 (Previous Year) (0.00) (12.9) (18.3) (31.2) Note 4 : Interest on non-performing assets is recognised on realisation basis as per the NHB Guidelines. Accordingly the total interest not recognised as at the Balance Sheet date is summarised as under:- Particulars Rs in millions Cummulative interest B/F from last Balance Sheet 56.6 Less : Recovered/ written back/ written off during the year 12.1 44.5 Add : Interest not recognised for the current year on - Sub Standard Assets 17.1 - Doubtful Assets 0.0 Total interest not recognised as on 31.03.2003 61.6 F- 48 Note 11: During the current year, consequent to OTS, the company has taken the vacant possession of mortgaged property consisting of Land & Building in Mumbai, in full satisfaction of Company’s claim as per the MOU/ Agreement at the valuation of Rs.35.0 millions. The shortfall in recovery amounting to Rs.8.0 millions has been adjusted in Books of Account as “write off”, with the requisite approvals. The property held as on year end has been shown as “other assets” in schedule 7. Note 13: With respect to company’s borrowing in FCNR(B)- Term Loan Account, the company has paid/ incurred interest amounting to Rs. 5.3 millions (Previous year Nil) in foreign currency. Further the company has taken a foreign currency fluctuation cover by way of Forward Exchange Contract from reputed approved dealer. Rs.6.2 millions being the liability of company with respect to exchange difference for the Forward Exchange Contract as on 31.03.2003 has been ascertained and accounted for as per the guidelines laid down by the Accounting Standard on accounting for the effects of changes in Foreign Exchange rates (AS-11) issued by The Institute of Chartered Accountants of India. Note 16: Contingent liabilities not provided for Rs in millions Particulars As at As at March 31, 2003 March 31, 2002 - Estimated amount of contracts remaining unexecuted (net of advance) 1.3 1.5 - Claim against the company not acknowledged as debt 0 0 - Income tax matters under dispute 43.3 62.8 - Interest tax matters under dispute 4.1 6.1 Year 2003-2004 Note 1 : The age-wise analysis of loans in respect of which the instalments (including interest) have become due are as under: - Rs in millions Particulars For the year Ended For the year Ended March 31, 2004 March 31, 2003 Outstanding for over six months 110.9 97.5 Outstanding for six months or less 93.0 57.9 203.9 155.4 F- 49 Note 3 : Provision for non-performing assets has been made in accordance with guidelines on prudential norms issued by National Housing Bank (NHB). Classification of loans and provisions made for non-performing assets is given hereunder: (Rs. in millions) Loans Standard Sub Standard Doubtful Total Housing Loans (including instalments due from borrowers) Individuals 7295.6 246.0 17.0 7558.6 (Previous Year) (6169.1) (163.4) (12.4) (6344.9) Corporate Bodies 132.1 0.00 58.7 190.8 (Previous Year) (12.1) (0.00) (60.3) (72.4) Other Loans 12.8 0.00 0.00 12.8 (Previous Year) (18.5) (0.00) (0.00) (18.5) Total Loans 7440.5 246.0 75.7 7762.2 (Previous Year) (6199.7) (163.4) (72.7) (6435.8) Provisions (as per NHB guidelines) 0.00 24.6 25.5 50.1 (Previous Year) (0.00) (16.3) (23.7) (40.0) Floating Provision (over and above 10.0 NHB Provisioning norms) (Previous Year) (0.00) Total Provisions 0.00 24.6 25.5 60.1 (Previous Year) (0.00) (16.3) (23.7) (40.0) Note 4 : Interest on non-performing assets is recognised on realisation basis as per the NHB Guidelines. Accordingly the total interest not recognised as at the Balance Sheet date is summarised as under:- Particulars Rs in millions Cumulative interest B/F from last Balance Sheet 61.6 Less: Recovered/ written back/ written off during the year 9.2 52.4 Add: Interest not recognised for the current year on - Sub Standard Assets 25.7 - Doubtful Assets 0.4 Total interest not recognised as on 31.03.2004 78.5 Note 11: During the previous year, the Company had taken the vacant possession of mortgaged property consisting of Land & Building in Mumbai, in full satisfaction of Company’s claim as per the MOU/ Agreement at the valuation of Rs.35.0 millions which has been shown as “Other Assets” in Schedule 7. Note 13: With respect to company’s borrowing in FCNR(B)- Term Loan Account and ECB, the company has paid/ incurred interest amounting to Rs.16.2 millions (Previous year Rs.5.3 millions) in foreign currency. Further the company has taken a foreign currency fluctuation cover by way of Forward Exchange Contract from reputed approved dealer. Rs.0.1 million (Previous year Rs.6.2 million) being the liability of company with respect to exchange difference for the Forward Exchange Contract as on 31.03.2004 has been ascertained and accounted for as per the guidelines laid down by the Accounting Standard on accounting for the effects of changes in Foreign Exchange rates (AS-11) issued by The Institute of Chartered Accountants of India. F- 50 Note 18: Contingent liabilities not provided for Rs in millions Particulars As at As at March 31, 2004 March 31, 2003 - Estimated amount of contracts remaining unexecuted (net of advance) 0.2 1.3 - Income tax matters under dispute 23.3 43.3 - Interest tax matters under dispute 4.1 4.1 NOTES ON ACCOUNTS 1. The age-wise analysis of loans in respect of which the instalments (including interest) have become due are as under: Rs in millions For the half year Ended For the year Ended September 30, 2004 March 31, 2004 Outstanding for over six months 136.2 110.9 Outstanding for six months or less 117.2 93.0 253.4 203.9 2. Housing loans and instalments due from borrowers shown under Loans and Advances are secured wholly or partly by: (a) Equitable Mortgage of Property (b) Pledge of shares, units, NSCs, other securities, assignment of life insurance policies. (c) Bank guarantees, corporate guarantees, government guarantee or personal guarantees. (d) Undertaking to create a security. 3. Provision for non-performing assets has been made in accordance with guidelines on prudential norms issued by National Housing Bank (NHB). Classification of loans and provisions made for non-performing assets is given hereunder: (Rs. in millions) Loans Standard Sub Standard Doubtful Total Housing Loans (including instalments due from borrowers) Individuals 7793.5 357.1 45.9 8196.5 (Previous Year) (7295.6) (246.0) (17.0) (7558.6) Corporate Bodies 208.3 0.00 58.7 267.0 (Previous Year) (132.1) (0.00) (58.7) (190.8) Other Loans 11.7 0.00 0.00 11.7 (Previous Year) (12.8) (0.00) (0.00) (12.8) Total Loans 8013.5 357.1 104.6 8475.2 (Previous Year) (7440.5) (246.0) (75.7) (7762.2) Provisions (as per NHB guidelines) 0.00 35.7 38.4 74.1 (Previous Year) (0.00) (24.6) (25.5) (50.1) Floating Provision (over and above 0.00 NHB Provisioning norms) (Previous Year) (10.0) Total Provisions 0.00 35.7 38.4 74.1 (Previous Year) (0.00) (24.6) (25.5) (60.1) F- 51 4. Interest on non-performing assets is recognised on realisation basis as per the NHB Guidelines. Accordingly the total interest not recognised as at the Balance Sheet date is summarised as under:- Rs in millions Cumulative interest B/F from last Balance Sheet 78.5 Less: Recovered/ written back/ written off during the half year - Add: Interest not recognised for the half year on - Sub Standard Assets (Net) 18.3 - Doubtful Assets 0 Total interest not recognised as on 30.09.2004 96.8 5. Managerial remuneration (paid in accordance with the regulations of Punjab National Bank):- Rs in millions For the half year Ended For the half year September 30, 2004 Ended March 31, 2004 Salaries and Allowances 0.2 0.3 Contribution to Provident Fund 0.0 0.0 Total 0.2 0.3 Value of perquisites (Car, Accommodation etc) 0.0 0.0 Travelling Expenses 0.1 0.2 6. Related Party Transactions: In view of the exemption available to the company under para 9 of Accounting Standard on Related Party Disclosures (AS-18), related party relationships with other state controlled enterprises and transactions with such enterprises are not being disclosed. However, the company has identified all other related parties having transactions during the year as given below: a) Key Management Personnel (Rs. in millions) S.No Name of transacting related party Amount 1 Sh. V. K. Sood & Relatives Salary & Allowances 0.0 (Managing Director joined on 17-10-2003. Assumed charge as Cont. to PF 0.0 MD on 16-12-2003 Resigned on 3-6-2004) Value of perquisites 0.0 Travelling exp. 0.0 2 Sh. K.G. Sathyasingan & Relatives Salary & Allowances 0.1 (Managing Director joined on 3-6-2004. Assumed charge Cont. to PF 0.0 as MD on 20-7-2004) Value of perquisites 0.0 Travelling exp. 0.1 b) Only the independent directors are paid sitting fees for the board / committee meetings at the rate of Rs.3000/- per meeting besides out of pocket expenses. During the year the Company has paid a sum of Rs.0.1 million towards sitting fees. F- 52 7. Deferred Taxes i) The break up of net deferred tax asset as at 30.9.2004 is as under: (Rs in millions) Deferred Tax Deferred Tax Asset Liability Timing Differences on account of : Differences between book depreciation and depreciation under the Income Tax Act, 1961 0.3 0 Interest not recognised on NPAs 29.4 0 Total 29.7 0 Net deferred tax asset 29.7 8. Earning per share (EPS) calculation (basic and diluted) Unit For Half Year 2003-04 ended 30.09.2004 a. Amount used as the numerator Profit after tax Rupees 77,479,547 154,965,052 b. Weighted average number of equity shares used as the denominator Number 30,000,000 30,000,000 c. Nominal value of shares Rupees 300,000,000 300,000,000 d. Earning per share (EPS) Rupees 2.58* 5.17 * not annualised 9. Segment Reporting : Company’s prime business is to provide loans for purchase, construction, repairs & renovations of Houses/ Flats etc. There are no business operations located “Outside India”. Hence all the activities are considered as a “ Single business/ Geographical Segment” for the purposes of Accounting Standard on Segment Reporting (AS-17), issued by The Institute of Chartered Accountants of India. 10. The provision for Income Tax has been made on the basis of the accounting practices consistently followed by the Company after allowing benefits under section 36(1)(viii) of the Income Tax Act, 1961. The method of bifurcation of income & expenses for long term housing finance is the same as that of last years’. 11. During the previous year, the Company had taken the vacant possession of mortgaged property consisting of Land & Building in Mumbai, in full satisfaction of Company’s claim as per the MOU/ Agreement at the valuation of Rs.35.0 millions which has been shown as “Other Assets” in Schedule 7. Profit or loss on sale of the property shall be ascertained and accounted for in the year of its disposal. 12. As per the information available with the company, there are no amounts payable to small scale industrial undertaking. 13. With respect to company’s borrowing in FCNR(B)- Term Loan Account and ECB, the company has paid/ incurred interest amounting to Rs.8.5 millions (Previous year Rs. 16.2 millions) in foreign currency. Further the company has taken a foreign currency fluctuation cover by way of Forward Exchange Contract from reputed approved dealer. Rs.0.5 million (Previous year Rs.0.1 million) being the liability of company with respect to exchange difference for the Forward Exchange Contract as on 30.09.2004 has been ascertained and accounted for as per the guidelines laid down by the Accounting Standard on accounting for the effects of changes in Foreign Exchange rates (AS-11) issued by The Institute of Chartered Accountants of India. 14. All Computer Software purchased from outsiders are being amortised as per Accounting Standard 26 (AS-26) as laid down by The Institute of Chartered Accountants of India. Considering the technological risks and issues of technological obsolescence, the Depreciation at the rate of 60% p.a. is being provided. All subsequent expenditure on software after its purchase and /or installation are recognised as expenditure whenever incurred. F- 53 15. Government Notification about Cess payable under section u/s 441-A of the Companies Act, 1956, being not available, necessary provision has not yet been made. 16. Amount due from officers as at 30.9.2004 was Rs.1,100/- (Previous year Rs.4,400/-). Maximum amount due from officers at any time during the year was Rs.4,400 /- (Previous year Rs. 5,500/-). 17. Contingent liabilities not provided for Rs in millions As at As at September 30, 2004 March 31, 2004 - Estimated amount of contracts remaining unexecuted (net of advance) 0.1 0.2 - Claims against the Company not acknowledged as debt 0.1 0.0 - Income tax matters under dispute 26.2 23.3 - Interest tax matters under dispute 4.1 4.1 19. Previous year figures have been rearranged/regrouped wherever necessary. 20. Figures have been rounded off to the nearest rupee. 21. BALANCE SHEET ABSTRACT AND COMPANY’S GENERAL BUSINESS PROFILE I. REGISTRATION DETAILS Registration No. 55-33856 State Code 55 Balance Sheet Date 30.09.2004 II. CAPITAL RAISED DURING THE YEAR (Amount in Rs. millions) Public Issue NIL Right issue NIL Bonus Issue NIL Private Placement NIL III. POSITION OF MOBILISATION AND DEPLOYMENT OF FUNDS (Amount in Rs. millions) Total Liabilities 8296.6 Total Assets 8296.6 SOURCE OF FUNDS Paid up Capital 300.0 Reserves & Surplus 653.3 Secured Loans 5880.0 Unsecured Loans 1463.3 APPLICATION OF FUNDS Net Fixed Assets 29.5 Investments 423.7 Net Current Assets 7843.4 Miscellaneous Expenditure 0 Accumulated Losses 0 IV. PERFORMANCE OF THE COMPANY Amount in Rs. millions) Turnover and other income 430.4 Total Expenditure 332.8 Profit before tax 97.6 Profit After Tax 77.5 Earning per share (rupees) 2.58 Dividend Rate (%) 0 V. GENERIC NAME OF THREE PRINCIPAL PRODUCTS/SERVICES OF COMPANY (As per Monetary terms) Item code No. N.A. Product Description Housing Finance Activities F- 54 ANNEXURE- E GUPTA GOEL & KHANNA Chartered Accountants 68, DARYAGANJ, NEW DELHI 110002 Ph.23275830, 35214455 Fax: 23260007 The Board of Directors PNB Asset Management Company Limited 4th floor, PNB Building 5 Sansad Marg, New Delhi-110001 Sir, As desired for the purpose of certification of statement of accounts to be incorporated in the offer document proposed to be issued by Punjab National Bank in connection with the follow on public offer of the Equity Shares, We stated as follows:- 1. We have examined the audited financial statement of PNB Asset Management Company Ltd., for the five financial year ending with 31st March, 2004 being the last date up to which accounts have been made and audited by the auditors of the company of those respective years and unaudited financial statement for the half year ending 30th September, 2004. We certify that the financial statement for the six months ending 30th September, 2004 has not been subjected by us to a complete audit in accordance with the generally accepted auditing practices necessary for expressing an opinion whether the statement show a true and fair view. 2. The aforesaid financial statement have been prepare in accordance with the Companies Act, 1956 and as per SEBI Regulation issued from time to time and subject to the limitation for disclosure required therein. We however, conducted a limited review of the six months ending on 30th September, 2004 to obtain moderate assurance as to whether the financial statements are free of material misstatement. A review is limited primarily to the enquiries of the Company personnel and procedures applied to financial data and thus provide less assurance than audit. We have not performed an audit and accordingly, we do not express an opinion on the same. 3. The profit & Loss for the five financial years ending on 31st March, 2004 and the Balance Sheet as at the end of the respective years and the significant accounting policies are prepared from the aforesaid accounts and making such adjustments and regrouping which were in our opinion considered appropriate, subjects to notes on accounts as per audit reports of respective years and various qualifications made in the audit’s report of respective years. Based on the limited review for the six months ending 30th September, 2004 conducted by us as aforesaid, nothing has come to our notice that causes us to believe that the accompanying statements do not give a true and fair view in accordance with the accounting standards, other recognized accounting policies and practices and relevant statutory requirements. The figures of five years have been complied and verified from the audited statement of account of the company. For Gupta Goel & Khanna Chartered Accountants Vinod Khanna Partner M.no.81148 New Delhi 15.10.2004 F- 55 PNB ASSETS MANAGEMENT CO. LTD I. SUMMARIZED PROFIT AND LOSS ACCOUNT Rs. In millions Audited Reviewed Year ended March 31 2000 2001 2002 2003 2004 30.9.04 INCOME Management fees 26.7 19.0 13.6 14.1 15.9 1.1 Dividend 0.4 0.1 1.9 1.6 1.5 0.2 Interest 17.0 17.8 17.4 15.1 13.2 6.2 Profit on sale of Inv. 9.6 0.8 7.8 1.1 2.8 0.0 Profit on sale of Assets 0.0 0.0 - - - 0.0 Int. on Income tax refund 0.4 0.5 0.5 0.3 - 0.3 Miscellaneous Income 0.0 0.1 0.5 0.0 0.0 32.0 Provision for expenses written back 0.1 0.0 0.0 0.1 0.0 0.1 Provision for fall in value of Inv. Written back 0.0 0.0 0.0 3.0 6.8 - TOTAL INCOME 54.2 38.3 41.7 35.3 40.2 39.9 EXPENDITURE Employees remuneration & benefits 5.5 6.4 6.1 6.1 6.8 4.5 Legal & Prof. Charges 0.5 1.6 1.1 1.0 0.9 0.1 Security Expenses 0.1 0.1 0.1 0.1 0.1 0.0 Rent 3.0 2.0 2.0 2.0 2.0 0.0 Electricity & Water charges 0.1 0.0 0.0 0.0 0.0 - Insurance 0.0 0.3 0.0 0.0 0.1 0.3 Communications 1.4 1.5 1.4 1.4 1.3 0.3 Auditor’s fees 0.1 0.0 0.1 0.1 0.3 0.1 Printing & Stationery 0.2 0.3 0.3 0.3 0.3 0.1 Repairs & Maintenance 0.5 0.5 0.7 0.6 0.8 0.1 Travelling & Conveyance 1.2 1.1 1.4 1.7 1.3 0.6 Business Promotion 0.0 0.0 0.2 0.2 0.1 0.0 Entertainment 0.2 0.3 0.2 0.3 0.1 - Initial Issue Exp. Amortised 0.2 0.2 0.2 0.2 0 - F- 56 Rs. In millions Audited Reviewed Year ended March 31 2000 2001 2002 2003 2004 30.9.04 Miscellaneous Exp. 0.2 0.1 0.1 0.1 1.2 0.3 Scheme mgmt. Exp. A) on account of assured returns 52.1 22 8.5 0 0 - B) others 6.4 1 1.1 0.6 1.2 0.4 Depreciation 0.8 0.9 1.5 1.4 1.4 0.6 Loss on sale of fixed Assets 0.2 0.1 0 0 0 - Loss on sale of inv. 0.3 1.1 0.1 0.1 3.7 - TOTAL 73.0 39.5 25.1 16.2 21.6 7.4 Profit/(loss) for the yr. Before tax -18.8 -1.2 16.6 19.1 18.6 32.5 App./Dep. In investment 2.0 -7.1 0.0 0.0 0.0 -1.3 Less prior period adj. 0.0 0.9 -0.5 0.1 0.0 0.0 Less short prov. Of Income 0.0 0.0 0.0 0.0 0.0 0.0 Tax for earlier years 0.0 0.0 0.0 0.0 0.0 - Profit/loss before tax -16.8 -7.4 16.1 19.2 18.6 31.2 Prov. For tax 0.0 0.0 1.1 1.7 1.1 0.7 Profit/loss after tax -16.8 -7.4 15.0 17.5 17.5 30.5 Profit/loss brought forward -97.5 -114.4 -121.7 -106.7 -89.2 -71.7 Profit/loss available for appropriation -114.3 -121.8 106.7 -89.2 -71.7 -41.2 F- 57 II. SUMMARIZED STATEMENT OF ASSETS AND LIABILITIES Rs. in millions Audited Reviewed AS AT MARCH 31 2000 2001 2002 2003 2004 30.9.04 LIABILITIES Share capital 290.0 290.0 290.0 290. 290.0 290.0 Reserves & Surplus 0.0 0.0 0.0 0.0 0.0 0.0 Profit & Loss Account 0.0 0.0 0.0 0.0 0.0 0.0 Current Liabilities 2.3 4.8 1.8 0.9 1.3 0.2 Provisions 0.0 0.0 0.0 0.0 0.0 - A) for assured returns 13.6 10.9 0.0 0.0 0.0 - B) others 0.2 0.1 1.2 3 4.3 2.3 TOTAL LIABILITES 306.1 305.8 293.0 293.9 295.6 292.5 ASSETS Fixed Assets (net of dep.) 2.6 8.2 6.3 8.0 6.7 6.0 Investments 114.9 119.3 32.7 28.4 35.1 24.6 Interest/div.Accrued 3.7 6.6 3.8 1.7 2.2 4.5 Cash & Bank balance 23.6 39.6 135.3 156.4 149.4 208.1 Loans & Advances 16.5 8.5 7.6 8.6 21.8 8.1 Prepaid Expenses 0.3 0.3 0.1 0.1 0.1 0.0 Interest Recoverable 4.3 0.9 0.1 0.0 0.3 - Other current assets (including sundry debtors) 25.3 0.3 0.2 1.5 8.3 - Preliminary/Preop. exps. (to the extent written off) 0.5 0.4 0.2 0.0 0.0 - Profit & loss account 114.4 121.7 106.7 89.2 71.7 41.2 TOTAL ASSETS 306.1 305.8 293.0 293.9 295.6 292.5 III. No adjustments resulting from Audit Qualifications, changes in accounting policies and other matters are required. F- 58 IV SIGNIFICANT ACCOUNTING POLICIES 1. (a) Basis of Accounting The financial statements are prepared under the historical cost convention on accrual basis and are in accordance with the requirements of the Companies Act, 1956. (b) Fixed Assets Fixed Assets are stated at cost of acquisition. Depreciation is provided on straight line method at the rates not less than as specified in Schedule XIV of the Companies Act, 1956. (c) Investments (a) Long-term investments of the Company are intended to be held till maturity. However Govt. Securities/PSU Bonds/Equity Shares may be sold even before maturity keeping in view the market conditions, interest scenario or as per requirements. (a) Investments are stated at cost on the date of Balance Sheet. However, provision is made for diminution in the value of investments as per market rates and on YTM basis for non-traded debt on the date of Balance Sheet. Appreciation, if any, has been ignored. (b) NCDs bought from PP 91, RIPS 90 (Rollover) and RIPS 94(schemes of PNB Mutual Fund) were valued at cost on the date of Balance Sheet as on 31.3.2002 and no provision was made for the diminution in the value of these NCDs. As per the purchase agreement, net shortfall, if any, either of Principal at the time of realisation of these securities or of interest may be met out of future realisations/income earned in respective schemes. 2. REVENUE RECOGNITION (a) Revenue is recognised on accrual basis as per the recognised mercantile system of Accounting. However, the accrual of interest in the default debentures is not being done. Income in these cases is being recognised on actual receipt basis. (b) Management fee is recognised on accrual basis as certified by the auditors of PNB Mutual Fund and as per the rates agreed between Board of Trustees and PNB Asset Management Company Ltd. in terms of SEBI regulations. 3. PRELIMINARY EXPENSES Preliminary expenses incurred on incorporation of the Company are amortised over a period of ten years in equal instalments. Expenses incurred post incorporation are being amortised over a period of five years in equal instalments. 4. EXPENSES OF SCHEMES MANAGEMENT Initial issue cost/expenses under each scheme exceeding prescribed limits as per SEBI (Mutual Funds) Regulations, 1996 and Asset Management Company agreement with PNB Mutual Fund, if any, are absorbed as revenue expenditure in the year in which the same are incurred. 5. RETIREMENT BENEFITS (a) Valuation of Leave Encashment is based on actuarial valuation. (b) The Company has taken the policy of Life Insurance Corporation under Group Gratuity Scheme to cover the liability for gratuity for its employees only and not for the employees on deputation from PNB. F- 59 V SIGNIFICANT CHANGES IN ACCOUNTING POLICIES Changes between Ist April, 1999 and 30th September, 2004 - NIL – However, during the year financial year 2001-2002, the rates of Depreciation have been revised. The category wise changes in the rates of depreciation are given below: Category Previous Rates Revised/ Current rates Furniture and Fittings 6.33% 10.00% Vehicles 9.50% 20.00% EDP 16.21% 33.33% Equipment 6.33% 6.33% Software - 20.00 VI. MATERIALNOTES ON ACCOUNTS FOR THE PERIOD 1.4.99 TO 30.09.2004 1. Contingent Liabilities as on 30.09.2004 –NIL 2. The Company being an Asset Management Company of PNB Mutual Fund, the main source of income is the management fee charged from various schemes floated by PNB Mutual Fund. Simultaneously the company is earning interest / dividend on funds invested in Bank FDRs, debt and equity. 3. The Managing Director-Shri Ranjan Dhawan was on deputation from Punjab National Bank till 05/07/03. After 05/07/03 Managing Director Shri B B Aggarwal is on Deputation from Punjab National Bank and remuneration of both is in accordance with the service rules of the Bank. Perquisites have been calculated as per the Income Tax Act amended up to date. 4. Regulation 52(6) of SEBI Mutual Funds regulations provide that total annual recurring expenses and management fees including additional fee shall at all time be within the limits prescribed under the regulations. The AMC is required to bear the excess recurring expenditure. Accordingly the excess recurring expenditure of Rs.250,000/- of BGF-99 scheme have been borne by AMC in the financial year ended 2004 though provision of Rs. 300,000/- was made in the books of the AMC. For the half year ended Sep. 2004 the excess expenditure borne by the AMC is Rs. 56,200/- Initial issue expenses for BGF 99 and Debt Fund 99 were incurred to the tune of Rs.265,908/- and Rs. 5,354,992/- respectively in the financial year ending March 31, 2000. As per the offer document AMC is entitled to charge an additional management fee upto 1% of weekly average net assets till the initial issue expenses borne by AMC have been recovered. In case the fund introduces an entry load at a later stage, the additional management fee will be charged only for/till the time no entry load is imposed on sale of units. 5. The company was managing four schemes of PNB Mutual Fund and NAV of all the schemes are above par as on 31.3.2004. The net worth of the company as on 31.3.2004 is Rs.218.3 millions against paid up capital of Rs.290 millions. 6. Legal and professional charges includes professional charges and legal charges on account of suits filed by the Company on behalf of Mutual Fund. 7. Actuarial Liability of leave encashment upto 31.03.2004 was assessed at Rs.260,392/-. The total liability has been fully provided for. However, the Company has taken a policy from Life Insurance Corporation of India for Group Gratuity Scheme and had paid a sum of Rs. 55,452/- as premium of this Group Gratuity Scheme. 8. Leave Travel Concession ( LTC) benefits payable to the employees are being accounted on cash basis. 9. (a) Rent, at the prevailing market rate, amounting to Rs.1,986,600/- was paid to PNB for the year 2003-04 on account of office premises. (a) Bank accounts are being maintained mainly with PNB for carrying out day to day operations and services of PNB GILTS have been utilised for transactions/custody of Government securities. 10. Gross interest for Rs.13.25 million shown as other income (Schedule 11) includes Interest on deposits Rs.11.09 million and Interest on investment Rs.2.15 million, however these figures are inclusive of deductions of income tax amounting to Rs.2.36 million and Rs.0.04 million respectively. F- 60 11. The year-wise breakup of TDS (Schedule 7) is as under: Assessment year Amount 2000-2001 2,587,096 2001-2002 54,197 2002-2003 47,147 2003-2004 81,408 2004-2005 3,152,052 Total 5,921,900 While the assessment for the year 2004-05 is yet to be done by the income-tax authorities, assessments for the earlier years have been completed and the refunds are pending for want of certain formalities. 12. As per the requirements of Accounting Standard 22 (AS-22) “Accounting for Taxes on Income” which became applicable w.e.f. 1.04.2002, the difference between written down value of Fixed Assets as per Financial Accounts & as per the Income Tax Act 1961 deferred tax liability of Rs.0.5 million and Rs. 195,593/- for the year ending 31.3.2003 and 31.3.2004 respectively have been transferred from Profit & Loss A/c to Deferred Tax Liability. 13. Provision for Income Tax has been made as per Minimum Alternate Tax. 14. On 30th April 2004 all the schemes of PNB Mutual Fund have been transferred to Principal Financial Group. Now PNB Asset Management Company Limited is to be merged back in its holding Company Punjab National Bank. As per business transfer agreement, the company has received retirement fee of Rs. 3.20 Crores. 15. Miscellaneous expenses for the half year ended 30.9.2004 includes Rs. 200,000/- as printing charges and Rs. 436,416/- as postage charges for sending 76500 letters to all scheme holders for giving information about transfer of schemes to Principal Financial Group on 30/4/04. 16. Consequent on application of Accounting Standard 26 – Intangible Assets (AS-26) prescribed by Institute of Chartered Accountant of India which became applicable from 01/04/03: - All Intangible items should be written off against opening Revenue Reserves, but due to non-availability of Reserves, Intangible items amounting to Rs. 42901/- cannot be written off, so these items are written off by debiting P&L A/c in usual manner. 17. No provision for CESS on turnover under section 441A of the Companies Act 1956 (as amended by Second Amendment Act 2002) has been made in the books of account in the absence of any notification regarding the applicable tax rate on the company. However, the maximum liability could be of Rs.33, 375/-. 18. Return of 12.5% was assured under the PNB Regular Income Plus Scheme-1990(Rollover). The distribution of the return is done on 31st August every year. The amount of shortfall borne for the year ended 31.8.2001 was Rs.30,443,515/-. The scheme was matured on 31.8.2001. 19. NCDs of RIPS 90(Rollover) were purchased by company during the year ending March 2002 amounting to Rs.1.66 millions on yield to maturing basis for arranging immediate liquidity for redemption of the scheme. 20. NCDS of RIPS 94 were purchased by the company during the year ending March 2002 amounting to Rs.2.32 millions on yield to maturity basis for arranging immediate liquidity for redemption of the scheme. 21. NCDs of PP 91 were purchased by AMC at Rs.33.1 millions on yield to maturity basis for arranging immediate liquidity for redemption of the scheme during the financial year 1999-2000. As per the purchase agreement, the net shortfall (if any) at the time of realisation/redemption of these NCDs (i.e.overall realisation below Rs.33.1millions) is to be met by future realisations/incomes in respect of pp-91 redemption fund under the trust. As such provision for any diminution in value of these NCDs is not being accounted for and these securities are being valued at cost. The shortfall has since been recovered from the scheme as per the scheme of arrangement approved by the Board of Trustees and Board of AMC. 22. AS of March 31 2002 2003 2004 30.9.2004 Earning per share(Rs.) 0.52 0.60. 0.61 1.05 Net Asset Value per Share(Rs.) 6.32 6.92 7.53 8.37 F- 61 PNB CAPITAL SERVICES LIMITED ANNEXURE - F SUMMARIZED PROFIT & LOSS ACCOUNT ACCOUNT Rs. in million 2000 2001 2002 2003 INCOME i. Lease and Hire Purchase 66.7 38.0 56.3 3.0 ii. Investments 49.8 44.6 17.4 (8.4) iii. Non Fund Based 13.6 10.2 5.2 7.9 iv. Others Interest On Deposits 1.5 0.5 0.1 2.7 Miscelleneous Income 8.5 7.7 21.9 41.9 Provision No Longer Required 17.0 26.9 52.7 0.0 Profit on sale of assets 0.0 0.0 0.0 0.0 TOTAL 157.1 127.9 153.6 47.1 EXPENDITURE Personnel & other administrative expenses 20.0 14.6 9.8 5.6 Provision and other expenses 68.6 47.0 79.1 0.6 Intt on deposits and borrowings 95.3 54.6 23.2 0.3 TOTAL 183.9 116.2 112.1 6.5 PBDT (26.8) 11.7 41.5 40.6 Less Depreciation (31.8) 25.9 10.1 0.9 PBT (58.6) (14.2) 31.4 39.7 Provison for Tax 0.0 0.0 2.2 8.3 TDS Certificate Not Acknowledged 0.0 0.0 0.2 0.0 PAT (58.6) (14.2) 29.0 31.4 Less: Tax for previous years provided/ paid (0.1) 0.0 0.0 0.0 Add: Excess provision for tax written back 0.9 0.0 0.0 0.0 Profit/ (Loss) (57.8) (14.2) 29.0 31.4 Loss Appropriated from General Reserve 0.0 0.0 0.0 0.0 Profit/ (Loss) carried over to Balance Sheet (57.8) (14.2) 29.0 31.4 Note: Merged with Punjab National Bank with effect from 1st April 2003 F- 62 SUMMARIZED STATEMENT OF ASSETS AND LIABILITIES Rs. in millions AS AT MARCH 31 2000 2001 2002 2003 LIABILITIES Capital 200.0 200.0 200.0 200.0 Reserves & Surplus 15.0 15.0 15.0 15.0 Secured Loans 128.7 54.8 25.4 0.0 Unsecured Loans 471.8 243.1 0.0 0.0 TOTAL 815.5 512.9 240.4 215.0 APPLICATION OF FUNDS FIXED ASSETS Gross Block 1,376.5 1,347.0 951.7 337.1 Less: Depreciation 1,150.8 1,209.5 868.3 264.7 Net Block 225.7 137.5 83.4 72.4 INVESTMENTS 565.4 392.1 250.4 191.4 Less: Depreciation 173.6 176.3 183.4 153.4 Net Investments 391.8 215.8 67.0 38.0 CURRENT ASSETS, LOANS & ADVANCES Inventory 24.4 9.5 0.0 0.0 Sundry Debtors 62.3 79.6 20.5 0.5 Loans & Advances 50.6 66.0 63.9 63.8 Cash & Bank Balances 2.1 5.0 13.0 80.2 Other Current Assets 126.6 61.2 26.1 5.3 Total 266.0 221.3 123.5 149.8 LESS: CURRENT LIABILITIES & PROVISIONS Current Liabilities 93.4 70.7 14.5 4.6 Provisions 51.9 82.6 81.4 71.7 Total 145.3 153.3 95.9 76.3 Net Current Assets 120.7 68.1 27.5 73.5 Profit & Loss Account (Dr Balance) 77.3 91.5 62.5 31.1 TOTAL 815.5 512.9 240.4 215.0 Note: Merged with Punjab National Bank with effect from 1st April 2003 F- 63 SIGNIFICANT ACCOUNTING POLICIES (2002/2003) 1. Accounting Convention The accounts have been drawn up on the historical cost convention conforming to the statutory provisions and prevailing practices. Revenue, costs, rights and liabilities are recognized as they are earned or incurred and recorded in the financial statements of the period to which they relate. 2. Revenue Recognition Income remaining unrealized as at the end of the year for more than stipulated period in terms of the NBFC Prudential Norms (RBI) Directions, 1998 issued by Reserve Bank Of India on 31.1.98 has not been organized. In compliance thereof income from NPA/ sub standard assets is recognized on receipt basis. Subject to above, income under various heads is recognized as follows: i) Merchant Banking: a) Issue Management Fee: Income is considered in full wherever the Public/ Rights issues are closed for subscription on or before the year-end. Non-refundable advance is recognized as income upon receipt. b) Underwriting Commission Income is considered on the basis of minimum underwriting commission upon opening of the issue; wherever subscription list has closed before the year-end and there is no devolvement income is recognized in full. ii) Leasing Finance Primary Lease Rentals are accounted for on accrual basis and secondary lease rentals are accounted for on receipt basis. Assets purchased and given on lease, which do not require erection, are capitalized at cost upon confirmation of delivery. Assets requiring installations/ erection are capitalized on receipt of confirmation of installation/ erection from the lessee. Lease rentals are considered from the date of capitalization based on a constant prevailing rate of return on the company’s investment outstanding on the lease. Finance charges accrue for intervening period on related advances till capitalization of leased assets. The interest on overdue lease rentals has been accounted for as Income when the collection for the same is reasonably certain. Lease Management Fee is recovered in full in the year in which lease agreements are executed. This is recognized as income in the year of receipt. iii) Hire Purchase Hire charges on Hire Purchase transactions due during the year are taken to Income generally on Equal Spread method and Capital Recovery method as per specific terms of agreement. iv) Intercorporate Deposit Income on ICD is accounted for on accrual basis on the amount of deposits outstanding. v) Trusteeship Fees Acceptance fee is considered as income in full in the year of acceptance of Trusteeship. Annual Trust Management Fee is taken as income on accrual basis from the date of allotment of securities. vi) Equity Parking Service charges on funds deployed on Equity parking are taken as income on accrual basis. vii) Project Appraisal Income relating to project appraisals is considered in two stages, 25% on acceptance of the assignment and 75% upon completion of the appraisal. F- 64 viii) Mergers and Acquisitions Income is recognized on completion of assignment or on receipt of fees which ever is earlier. ix) Placement of Shares / Debentures/ Bonds Income is recognized on completion of assignment or on receipt of fees which ever is earlier. 3. Fixed Assets Fixed Assets including assets given on lease are stated at historical cost, inclusive of installation charges, net of depreciation and lease equalization charge. 4. Depreciation Depreciation on fixed assets other than leased assets is provided on return down value method at rates prescribed in schedule XIV of the Companies Act. Depreciation on leased assets is provided on straight-line method at the rates prescribed in schedule XIV of the Companies Act. Pro rata depreciation is considered on assets acquired/ disposed off during the year 5) Stock on Hire Stock on Hire represents the cost of hire purchase assets less margin contributed by the hirer and installments falling due during the year. 6) Investments Investments, which are intended to be held as per permanent investments, i.e on long term basis or till maturity, are valued at costs, except in case of permanent diminution in value. Investments are capitalized at cost plus brokerage and stamp charges. Current Investments are valued at lower of cost or market value (Net of Expenses necessarily to be incurred on or before disclosure) for each investment, individually. Suitable provisions are made as per RBI Guidelines in case of doubtful/ disputed investments. All those listed securities, which have not been quoted for substantial period, have been considered as “Unquoted” Investments. “Unquoted” Investments are valued as under; (a) Equity Shares – At Cost or Break up value of the share as per the last audited balance sheet of the company concerned whichever is less. Incase the latest Balance Sheet is not available; the shares are valued at Rs. 1/- per company. (b) Preference Share – at cost or ace Value which ever is less. (c) Debentures – at carrying cost if interest thereon is received regularly, where interest servicing is not regular, depending on the tenor they are treated as long term loans or other credit facilities for the purpose of income recognition and asset classification. On the basis of methods of valuation as stated above, the resultant figure of depreciation (class- wise) is charged to the profit and loss account; and appreciation, if any ignored. The NPA guidelines are also applied and provisions if any, are charged to profit and loss account. 7. Categorization of / provisioning for loans, advances, investments etc: As per RBI guidelines NBFC Prudential Norms (RBI) Directions, 1998 as revised from time to time, loans, advances, investments, debts and other amounts recoverable are categorized into standard, sub standard, doubtful and loss assets, and provision based F- 65 on a percentage thereof is made other than for standard assets as under: Categorization Provisions (a) Sub Standard - 10% (b) Doubtful Assets 100% on unsecured portion 20% - 50% on the secured portion depending on the age of the non-performing assets. (c ) Loss Assets 100% Provision for Lease Assets In term of the RBI Guidelines, Provisioning for lease/ Hire purchase assets has been made as follows Categorization Provisions * If lease rental / Hire Purchase are over due for - More than 12 months but upto 24 months 10% of the net book value - More than 24 months but upto 36 months 40% of the net book value - More than 36 months but upto 48 months 70 % of the net book value - More than 48 months 100 % of the net book value * In addition to 100% provision of the amount of overdue net lease rentals taken to the credit of P& L Accounts in the earlier years. 8) Retirement Benefits The company has only Gratuity and PF as retirement benefits. Provision for Gratuity Liability in respect of eligible employees who are recruited by the company is made on the basis of actuarial valuation and policy of the same has been taken with LIC and premium paid. Company’s contribution to PF has been charged to Profit and loss Account. Regarding Employees on deputation from Punjab National Bank, the retirement benefit accrue and are accounted for at Punjab National Bank. F- 66 Notes forming part of accounts (2002/2003) 1. There are contingent liabilities on a/c of Rs in million 31.03.03 31.03.02 A) Bank Guarantee in respect of Lease / HP Finance 0.100 0.100 B) Claim against company not acknowledged as Debts 0.675 0.675 c) Demand raised by sales tax authorities which are under appeal 0.609 2.00 2. The sales tax levied by various governments on Leased transactions is recoverable form the lessee. Whatever sales tax recovered by the company has been deposited as per rule. However, there is certain demand amounting to Rs. 0.609 million raised by Sales Tax authorities of UP which are under dispute and appeals for revision / rectification have been filed. No liability can be ascertained at this stage. 3. Managerial Remuneration a. The Managing Director is on deputation from Punjab National Bank and her remuneration is in accordance with the service rule of the bank: (Break up of remuneration is as follows) (Rs in million) 31.03.03 31.03.02 Salary 0.336 0.303 Contribution to PF 0.022 0.022 Other Perquisites 0.00 0.048 A Chauffeur driven car has been provided to the Managing Director for official use. 31.03.03 31.03.02 Directors sitting Fees 0.018 0.003 Directors Traveling Fees 0.099 0.025 4.a The company’s investment in shares, bonds and Government Securities aggregate to Rs. 202.9 million (Previous Year: 250.4 million). The company’s investment other than in the course of Merchant Banking business or devolvement of underwriting obligation aggregate to Rs. 78.0 million as on 31/03/2003 (Previous Year: 90.4 million), the details of which are shares Rs. 31.9 million, Bonds – Rs 7.0 million, Units- Rs. 0.7 million, NCDs/ FCDs – Rs. 38.4 million). 4.b Investment include shares having book value Rs. 239 Lacs (Previous – Rs. 23.9 million) Held under Equity Parking with buy back commitment by the contracting parties as per the terms of the respective agreements. Buy back is over due in four case (Previous year – 4 cases) and aggregate outstanding services charges is Rs. 25.2 million (Previous year – 25.2 million). Suits of specific performance have been filed in these cases. In the meantime depreciation have been provided in these investments as per RBI prudential norms. 4.c The company’s investment in shares of other body corporate aggregate to Rs. 146.2 million on 31/03/03 (Previous year – 179.6 million) which includes share worth 114.3 million due to devolvement in respect of underwriting obligation as part of Merchant banking activity of the company. As such, the company’s remaining investment in shares of Rs. 31.9 million is well within the levels stipulated in term of section 372 of the Companies Act 1956. However if the total figure of investment of Rs. 179.6 million is considered the same is beyond the limits specified under section 372 of the Companies Act 1956. 5. Disputed Demand of Tax a. Interest Tax The additions were made by the income tax authorities treating the interest on securities as chargeable interest under Interest Tax Act 1974 and the demand for Rs. 1964987.00 were raised related to the assessments years 1994 – 95 and 1995-96. The said demands have been adjusted against the refund due / payment made as per income tax returns filed by the company. F- 67 The above noted demands have been quashed by Commissioners of Income Tax (Appeals), however the department has filed the appeals before the tribunal and the said appeals are pending. Hence the alleged interest tax has neither been accounted for nor appropriated. b. Income Tax The demand of Rs. 38177707.00 has been made by the Income Tax Department for the assessment year 1994-95 has been paid by the company and the same are under appeal. Further, appeal against the demand of Rs. 200533722/ - made by the Income Tax partly. Hence the alleged demands have neither been accounted for nor appropriated. 6. The financial statements have been prepared on the basis of going concern concept. The company has however stopped taking any fresh fee based business also following the RBI’s advice to the sponsor bank to take steps to close the Company. Further, the sponsor bank has approved the draft scheme of amalgamation of the company with itself. 7. Balances shown under loan, advances, deposits, debtors and creditors are subject to the confirmation reconciliation and consequential adjustments. In the opinion of he management, the value of current assets, loans, and advances on realization in the ordinary course of business will not be less than the value at which these are stated in the Balance Sheet. 8. The company has no liabilities towards bonus payment as no employee is covered under he Payment of Bonus Act. 9. Public deposits Rs. 2145982/- (previous year Rs.7143322/-) including interest due there on are unclaimed deposits as on 31.3.2003. 10. Net value of Lease Assets for Rs. 71.7 million include assets of 71.7 million (previous year Rs.78.0 million) where the primary and secondary lease period are already over. Transfer of assets is pending due to non-receipt of request from the lessee/Nominee. 11. In compliance with the prudential norms, No income (last year Rs. nil million) has been derecognised during the year 2002-03. 12. Provisions for the Non-Performing Accounts (NPAs) have been made as per NBFC - Prudential Norm Reserve Bank Directions 1998 amended from time to time. 13. During the year, the company accepted one time settlement (OTS) in one lease finance and hire purchase accounts namely – Sumac International Ltd. in terms of its Compromise Policy, the payment as compromise approved is yet to be received by the company. 14. Chennai and Mumbai branches of the company were closed w.e.f 31st August 2002 and 31st December 2002 respectively. 15. There are unabsorbed losses brought forward from earlier years amounting to Rs. 14.5 million. Further, tax credit available to the company u/s 115JAAis Rs. 21.4 million. The provision for Income Tax has been made after taking into consideration the brought forward losses. 16. Reportable segments in respect of business operations of the company have been identified on the basis of nature of activities attached to each segment. The segment wise information is as under: Rs. In million Leasing & Hire Investments Non-fund base Others Total Purchase Segment Revenue 306.2 -844.5 789.9 4458.0 4709.6 Allocable expenses Not Available Not Available Not Available Not Available Not Available Segment Results 306.2 -844.5 789.9 4458.0 4709.6 Unallocable expenses - - - - 737.9 Segment Assets 7173.3 3806.6 NIL 15037.1 260173.0 Segment Liabilities Not Available Not Available Not Available Not Available 7630.4 17 Related Party Transactions As per Para 9 of the Accounting Standard 18 on Related Party Disclosures, the company being a state controlled enterprise is not required to make disclosures of related party relationships with other state controlled enterprises and transactions F- 68 with such enterprises. Other information as per standard is as under: The overall supervision and control of company vests with Board of Directors. All key managerial personnel including Managing Director of the company who are on the roll of the company are on deputation from Punjab National bank. They are working full time with the company. Details of managerial remuneration are disclosed vide Note No. 3 in Schedule ‘J’ above. Out of the total of Six Directors, One is independent director. Only the independent director is paid sitting fee for the Board/Committee meetings at the rate of Rs.3000 per meeting. 18 Accounting Standard 22 in regard to creation of deferred tax asset/liabilities is mandatory and became applicable to the company w.e.f 1.4.2001 and in accordance with the said Accounting Standard the calculation for accounting purposes of the deferred taxes asses are as under : As at As at 31.3.2003 31.3.2002 FIXED ASSETS As per Companies Act 72260620 83403056 As per Income Tax Act 74782936 105261470 Difference 2522316 21858414 LEASE RENT NOT RECOGNISED 21921611 25952905 Total 24446930 47811319 Tax Rate 36.75% 35.70% Tax Asset 8984215 17068641 Round off Rs. 8.984 million Rs.17.069 million However, on consideration of principle of prudence, reading with clauses 15 to 18 and various other clauses of the Accounting Standard 22, the deferred tax assets is not required to be created because there is no reasonable level of certainty supported by the evidences that the company shall have sufficient future taxable income on the realistic estimate of profit. Further, as per the instruction of RBI, company has stopped accepting new business. Hence, the said deferred tax assets for Rs. 8.984 million have not been raised and accounted for in the books of accounts. The same shall be created and accounted for as and when company has reasonable certainty about the profit. 19 In accordance with the Companies (Amendment) Act 2000, deleting the provisions relating to deemed public companies, the company made an application to the Registrar of Companies for modification in its Certificate of Incorporation. After getting the Certificate of Incorporation modified, the status of the company has become private limited. 20 Previous year figures have been regrouped/rearranged wherever necessary. F- 69 PNB SECURITIES LTD. ANNEXURE - G SUMMARIZED PROFIT & LOSS ACCOUNT (Rs. in million) Year ended March 31 2000 2001 16.08.01 A. INCOME i) Interest on FDR 0.4 0.3 0.1 B. EXPENDITURE i) Filing fee 0.0 0.0 0.0 ii) Audit fee 0.0 0.0 0.0 iii) Pre-Operative Exp. W/Off 0.0 0.0 0.0 iv) Preliminary Exp. W/Off 0.2 0.0 0.0 TOTAL 0.2 0.0 0.0 C. Profit/Loss before tax 0.2 0.3 0.1 Provision for tax 0.3 0.1 0.0 Income tax paid 0.0 0.1 0.0 Profit/Loss after tax (0.1) 0.1 0.1 Add:Opening balance 0.3 0.2 0.3 Balance Carried to Balance Sheet 0.2 0.3 0.4 STATEMENT OF ASSETS AND LIABILITIES Rs. in millions AS AT MARCH 31 2000 2001 16.8.01 LIABILITIES Share Capital 5.0 5.0 5.0 Reserves & Surplus (As per P/L a/c) 0.2 0.3 0.4 Current Liabilities & Provisions 0.5 0.2 0.1 Total 5.7 5.5 5.5 ASSETS Fixed assets 0.0 0.0 0.0 Cash in hand 0.0 0.0 0.0 Balance with Scheduled banks in 5.3 5.4 5.4 FDR 0.0 0.0 0.0 Balance with Scheduled bank in C/A 0.2 0.0 0.0 Intt. Accrued on FDR 0.2 0.0 0.0 Advance Tax paid 0.0 0.1 0.1 Misc expenditure (to the extent of not written off) 0.0 0.0 0.0 Total 5.7 5.5 5.5 Note: The company was liquidated on 16.8.2001 F- 70 SIGNIFICANT ACCOUNTING POLICIES - The Accounts of the company are prepared on accrual basis except otherwise stated in accordance with the normally accepted accounting principles. - The company has passed a special resolution in their 5th Annual General Meeting held on August 16, 2001 for members voluntary winding up u/s 484(1)(b) of the companies Act, 1956. Therefore the company has not followed going concern concept and has shown the assets and liabilities at net realizable value during the period under consideration. F- 71 Annexure-H I. STATEMENT OF CONSOLIDATED PROFIT AND LOSS (Rs. in million) Financial Year ended March 31 2002 2003 2004 A INCOME Interest Earned 68,025.8 76,751.1 79,721.2 Interest & Discount on advances/bills 33,619.3 37,732.8 39,466.0 Income on Investment 31,137.8 34,321.7 38,067.7 lnterest on balance with RBI and 2,276.3 1,793.8 1,092.1 other Inter-Bank Lending Interest on Income tax Others 992.4 2,902.8 1,095.4 OTHER INCOME 10,981.9 13,350.7 19,721.4 Commission, Exchange & Brokerage 4,396.9 4,857.7 5,577.8 Profit on sale of investments (Net) 5,667.6 7,731.0 13,506.3 Profit on revaluation of investments (Net) -581.7 -697.8 -1,171.2 Profit on sale of land,bldg & other assets (Net) 3.7 4.2 2.3 Profit on exchange transaction ( Net) 922.4 950.8 1,060.1 Miscelleneous Income 573.0 504.8 746.1 Total 79,007.7 90,101.8 99,442.6 B EXPENDITURE 1 Interest Expended 44,358.8 44,732.6 42,639.9 Interest on deposits 41,388.7 41,789.9 39,431.6 Interest on RBI /Inter-Bank borrowings 817.4 597.9 562.1 Others 2,152.7 2,344.8 2,646.2 Operating expenses 18,160.0 20,736.8 23,879.2 I Payment to & provision for employees 13,207.8 14,813.5 16,596.7 ii Amortisation of VRS Expenditure /goodwill - 124.4 373.3 iii Rent, Taxes & Lighting 950.5 1,066.0 1,209.4 iv Insurance 389.5 461.6 547.2 v Printing and Stationery 289.7 529.7 355.4 vi Advertisement 98.9 87.8 118.8 vii Postage, Telegrams, Telephones etc. 294.9 321.9 459.0 viii Repairs & Maintenance 196.4 206.8 250.3 ix Law Charges 79.1 87.8 134.1 x Director’s Fees, Allowances & expenses 3.8 3.8 3.8 xi Auditors’ fees & Expenses 133.9 156.4 179.9 xii Other Expenditure 1,638.3 1,691.4 2,189.7 xiii Depreciation on Banks Properties 877.2 1,185.7 1,461.6 TOTAL EXPENDITURE 62,518.8 65,469.4 66,519.1 F- 72 (Rs. in million) Financial Year ended March 31 2002 2003 2004 Gross Profit before provisions & Contingencies 16,488.9 24,632.4 32,923.5 Add: Share of earnings / lossess in associates 392.4 468.2 139.0 Less: Provisions & Contingencies 9,885.9 15,478.2 20,911.1 Consolidated Net Profit before minnorities’ interest 6,995.4 9,622.4 12,151.4 Less Minorities’ interest 280.4 226.7 171.6 Consolidated Profit attributable to Group 6,715.0 9,395.7 11,979.8 Add: Brought forward consolidated profit / loss attributable to group 25.3 908.6 1,447.4 6,740.3 10,304.3 13,427.2 APPROPRIATIONS Statutory Reserve 1,631.2 2,290.6 2,985.7 Revenue & Other Reserve 3,550.4 5,488.7 8,034.0 Intirim Dividend - - 663.3 Proposed Dividend 636.7 928.6 398.0 Dividend Tax 13.4 149.0 183.5 Balance carried over to Consolidated Balance Sheet 908.6 1,447.4 1,162.7 6,740.3 10,304.3 13,427.2 F- 73 II. STATEMENT OF CONSOLIDATED ASSETS AND LIABILITIES (Rs. in million) AS AT MARCH 31 2002 2003 2004 (A) ASSETS 1 Cash in Hand 7,311.4 6,290.6 6,995.7 2 Balance with RBI 43,706.7 59,399.5 60,433.6 3 Balance with Banks - In India 6,152.1 13,749.0 12,941.6 - Outside India 5,158.1 774.6 995.7 4 Money at call and Short Notice 1,818.3 764.2 6,970.4 5 Investments - In India 287,853.5 350,762.4 435,712.8 - Outside India 116.4 128.0 144.4 6 Advances - In India 347,451.7 407,981.3 478,622.8 - Outside India - - - 7 Fixed Assets 8,139.7 9,025.1 9,107.2 Less: Revaluation Reserve 3,381.3 3,295.8 3,210.4 4,758.4 5,729.3 5,896.8 8 Other Assets* 33,865.8 32,207.1 34,932.8 TOTAL (A) 738,192.40 877,786.0 1,043,646.6 * includes Rs.455.82Cr. Being unamortised VRS expenditure (B) LIABILITIES 1 Demand Deposits From Banks 1,112.5 1,660.2 1,431.8 Demand Deposits From others 66,466.1 97,178.5 97,602.2 2 Saving Deposits 216,642.0 256,479.0 304,226.1 3 Term Deposits From Banks 13,161.7 4,847.8 7,178.2 Term Deposits From others 345,234.3 399,351.8 470,295.5 4 Borrowings - In India 9,736.4 16,326.3 20,039.3 - Outside India 44.0 1,012.7 6,858.8 5 Other liabilities and Provisions 14,027.4 15,008.4 22,601.6 6 Subordinate Debts 37,584.6 44,040.7 60,603.0 TOTAL (B) 704,009.0 835,905.4 990,836.5 (C) NET ASSETS (C=A-B) 34,183.4 41,880.6 52,810.1 Represented by : (D) Share Capital 2,122.4 2,653.0 2,653.0 (E) Minority Interst 1,093.6 1,238.6 1,449.2 (F) Share Application money/Share Premium 1,644.9 - - (G) RESERVES AND SURPLUS I. Statutory Reserve 7,715.2 10,005.8 12,991.5 ii Capital Reserve a) Revaluation Reserve 3,381.3 3,295.8 3,210.4 b) Others 1,256.4 1,256.8 1,280.5 F- 74 (Rs. in million) AS AT MARCH 31 2002 2003 2004 Capital Reserve-on consolidation 581.1 581.1 581.1 iii Investment Fluctuation Reserve 3,101.3 5,271.3 10,279.8 iv. Share Premium - 1,114.3 1,114.3 v Revenue and other Reserve 15,759.9 18,312.3 21,298.0 vi Balance of Profit & Loss Account 908.6 1,447.4 1,162.7 Total 32,703.8 41,284.8 51,918.3 Less: Revaluation Reserve 3,381.3 3,295.8 3,210.4 TOTAL (G) 29,322.5 37,989.0 48,707.9 (H) TOTAL (C TO G) 34,183.4 41,880.6 52,810.1 III. CONTINGENT LIABILITIES (Rs. in million) AS AT MARCH 31 2002 2003 2004 i) Claims against the Bank not acknowledged as debt 2535.8 1861.7 2146.9 ii) Disputed income tax demand under appeal/ references etc. 3120.5 141.9 377.5 iii) Liability for partly paid investments 3.4 3.4 1.8 iv) Liability on account of outstanding forward exchange contracts 164314.5 163640.3 195434.7 v) Guarantees given on behalf of constituents 30989.6 35446.8 51265.2 vi) Acceptances,Endorcements and other Obligations 28081.9 33768.8 71679.4 vii) i) Other items for which the bank is contingently liable 221.3 1025.1 1395.6 TOTAL (I) 229,267.0 235,888.0 322,301.1 Bills for collection 29928.6 33347.6 48130.8 IV. CONSOLIDATED CASH FLOW STATEMENT (Rs.In Million) For the years ended 31st March 2002 2003 2004 A. Cash Flow from Operating Activities (i) Net Profit after Tax 6,603.0 9,154.3 12,012.4 Add Share of Earning in Associates 392.4 468.1 139.0 Net Profit before Minority’s Interest 6,995.4 9,622.4 12,151.4 Add Provision for Tax (net of deferred tax) 2,656.2 4,036.4 7,282.8 Profit before taxes ( i ) 9,651.6 13,658.8 19,434.2 (ii) Adjustment for : Depreciation Charges (Gross) 963.1 1,271.2 1,920.4 Less : Amount drawn from Revaluation Reserve (85.5) (85.5) (85.4) Provision for NPAs, Floating Provision towards NPAs 6,795.6 9,352.4 11,996.2 Provision on Standard Assets 605.3 1,211.1 475.0 Other Provisions (net) 628.9 287.1 1,417.0 Depreciation/ (Release) on Investments (net) (381.6) 1,862.9 (259.9) F- 75 (Rs.In Million) For the years ended 31st March 2002 2003 2004 Reserve utilised for accumulated loss on PNB Caps Taken Over 0.0 0.0 (16.1) Interest on Subordinate debts (Financing Activity) 1,529.8 1,723.8 1,951.9 Amortisation Expenses / Deferred Revenue Expenses/Issue Exp 1,199.4 1,291.6 (1,140.8) Profit / Loss on sale of Fixed Assets ( net ) (3.6) (4.0) (2.3) ( ii ) 11,251.4 16,910.6 16,256.0 Operating Profit before Changes in Operating Assets and Liabilities ( i+ii) 20,903.0 30,569.4 35,690.2 Adjustment for net change in Operating Assets and Liabilities Decrease / (Increase) in Investments (28,229.5) (60,077.0) (84,706.9) Decrease / (Increase) in Advances (73,440.3) (65,152.0) (74,707.7) Increase / (Decrease) in Deposits 79,756.5 116,900.6 121,216.5 Increase / (Decrease) in Borrowings 427.4 6,868.2 9,559.1 Decrease / (Increase) in Other Assets (2,891.5) (6,598.3) 1,484.0 Increase / (Decrease) in Other Liabilities & Provisions (1,037.2) (3,208.4) 7,351.0 (iii) (25,414.6) (11,266.9) (19,804.0) Cash generated from Operations ( i+ii+iii) (4,511.6) 19,302.5 15,886.2 Tax Paid (net of refund ) 1,649.4 (1,881.6) (10,351.7) Net Cash from Operating Activities ( A ) (2,862.2) 17,420.9 5,534.5 B. Cash Flow from Investing Activities Investments in Subsidiary Companies / JV / RRBs (10.0) 0.0 0.0 Purchase of Fixed Assets (net of Sales) (1,276.7) (2,277.5) (2,000.2) Net Cash used in investing Activities ( B ) (1,286.7) (2,277.5) (2,000.2) C. Cash Flow from Financing Activities Share Application Money Recd. 6,753.9 0.0 0.0 Issue Expenses (54.2) 0.0 0.0 Subordinate Bonds Issued 4,800.0 4,130.0 7,650.0 Redemption of Subordinate Bonds (1,899.8) 0.0 0.0 Interest paid on Subordinate Debts (1,529.8) (1,723.8) (1,951.9) Payments of Dividends / Corporate tax on Dividend (626.0) (718.4) (1,873.2) Net Cash from Financing Activities ( C ) 7,444.1 1,687.8 3,824.9 D Net Change in Cash and Cash Equivalents ( A+B+C ) 3,295.2 16,831.2 7,359.2 Cash and Cash Equivalents at the beginning of the year 60,851.4 64,146.6 80,977.8 Cash and Cash Equivalents at the end of the year 64,146.6 80,977.8 88,337.0 3,295.2 16,831.2 7,359.2 Note :- 1 All figures in bracket represent “Cash Outflow” 2 Direct taxes paid (net of refund) are treated as arising from operating activities and are not bifurcated between investing and financing activities. F- 76 V. SIGNIFICANT ACCOUNTING POLICIES AS ON 31ST MARCH 2004 Significant accounting policies adopted in preparation of these consolidated financial statements are set out below: 1. BASIS OF PREPARATION OF ACCOUNTS: The accompanying financial statements have been prepared to comply, in all material aspects, with applicable statutory regulatory provisions, Accounting Standards and generally accepted accounting principles and practices prevailing in India, except otherwise stated. 2. CONSOLIDATION PROCEDURE: 2.1 Consolidated financial statements of the group have been prepared on the basis of audited financial statements of Punjab National Bank (Parent) and its three subsidiaries namely (i) PNB Gilts Ltd, (ii) PNB Housing Finance Ltd., (iii) PNB Asset Management Co., Ltd and after eliminating intra group transactions unrealized profit / loss and making necessary adjustments wherever required to conform to the uniform accounting policies. The financial statements of the subsidiaries are drawn upto the same reporting date as that of parent i.e. 31st March 2004. 2.2 The differences between cost to the group of its investments in the subsidiary entities and the group’s portion of the equity of the subsidiaries is recognized in the financial statement as Goodwill / Capital Reserve. 2.3 Minority interest in the net assets of consolidated subsidiaries consists of: a) The amount of equity attributable to the minority at the date on which investments in a subsidiary is made. b) The minority share of movements in equity since date of parent subsidiary relationship came into existence. 2.4 Investments in associates, where the group holds more than 20% of equity, are accounted for using equity method. 3. TRANSACTIONS INVOLVING FOREIGN EXCHANGE 3.1 Monetary assets and liabilities, guarantees, acceptances, endorsements and other obligations are translated in Indian Rupee equivalent at the exchange rates prevailing at the end of the year as per Foreign Exchange Dealers’ Association of India (FEDAI) guidelines except (i) foreign currency deposits under various schemes and equivalent currency investments and other balances which are stated at notional rates, and (ii) advances of erstwhile London branches which are accounted for at the exchange rate prevailing on the date of parking in India. 3.2 Non-monetary items, other than fixed assets, are translated at exchange rate prevailing on the date of transaction. 3.3 Income and expenditure items are accounted for at the exchange rate prevailing on the date of transaction. 3.4 Forward exchange contracts are translated at the exchange rate prevailing on the date of commitment. Gain/loss on evaluation of outstanding forward exchange contracts is taken to revenue as per FEDAI guidelines. 4. INVESTMENTS The Investments are categorized and valued as per the applicable RBI norms. 5. ADVANCES 5.1 Advances are classified as performing and non-performing assets and provisions are made in accordance with prudential norms prescribed by RBI. 5.2 Advances are stated net of provisions and de-recognized/ suspended interest relating to non-performing assets. 6. FIXED ASSETS 6.1 Fixed assets are stated at cost except such premises, which have been stated at revalued price. 6.2 Depreciation on Fixed assets is provided for on straight-line method based on estimated life of the asset. Leasehold land and premises are amortized over the period of the lease. 6.3 Depreciation on the leased out assets is provided on straight-line method at the rates prescribed in schedule XIV to the Companies Act, 1956. 6.4 While depreciation on additions to assets is charged for full year, it is ignored in the year of sale/ disposal. In case of assets taken over from eNBL depreciation has been charged for the post taken over period. F- 77 6.5 (a) Cost of acquisition of Computer Software Systems is amortized over a period of its economic life, subject to a maximum upto 5 years, except software costing upto Rs.5000/-, is charged to revenue. (b) Goodwill arising on amalgamation of erstwhile Nedungadi Bank Ltd. is amortized over a period of 4 years upto 31st March 2003. The balance was written off in the year 2003-04. 7. LEASED ASSETS Provisions against income on assets given on lease are made as per prudential norms for leasing prescribed by RBI. 8. STAFF BENEFITS Expenses on Gratuity, Pension and Leave encashment are accounted for on accrual basis considering actuarial valuation. 9. AMORTISATION OF VRS EXPENDITURE Payments under Voluntary Retirement Scheme are amortized over a period of 5 years as per RBI guidelines, wherever applicable. 10. REVENUE RECOGNITION Income and expenditures are accounted for on accrual basis except Income on non-performing assets is recognised on realisation basis as per prudential norms prescribed by RBI. Commission, interest on overdue bills, exchange, locker rent, income from merchant banking transactions and dividend income on mutual fund and others are accounted for on realisation. Income from interest on refund of income tax and interest tax is accounted for in the year the order is passed by the concerned authority. Interest on matured term deposits for overdue period is accounted for as and when such deposits are renewed, as per Bank’s rules. The interest on overdue lease rentals has been accounted for as income when the collection of the same is reasonably certain. Income in respect of placement of shares/debentures/bonds is recognised on completion of assignment or on receipt of fees whichever is earlier. 11. LEASE RENTALS: Lease rentals are considered from the date of capitalization based on a constant prevailing rate of return on the group’s investments outstanding on the lease. Finance charges accrue for the intervening period on related advances till capitalization of leased assets. 12. HIRE PURCHASE Hire charges on Hire Purchase transactions due during the year are taken to income generally on equal spread method and capital recovery method as per specific terms of agreement. 13. TAXES ON INCOME Taxes on income for the year comprise of current tax liability and deferred tax which recognise, subject to the consideration of prudence in respect of deferred tax assets, timing differences, being the difference between taxable income and accounting income that originate in one period and are capable of reversal in one or more subsequent periods. VI. CHANGES IN ACCOUNTING POLICIES: 1. Goodwill arising out of amalgamation was earlier amortized over a period of 4 years. However, the parent has written off the balance amount of Rs.373.3 million in the year 2003-04, but for the said change, the profits would have been higher by Rs.248.9 million. 2. During the year 2002-03, bank has changed the accounting of Leave Encashment from Cash to accrual method. The liability ascertained on the basis of actuarial valuation amounting to Rs.765.9 million (net of Rs.250 million provided in previous year and Rs.51.9 million provision of eNBL) pertaining to the year upto 31st March 2002 has been charged to Revenue and Other Reserve. Liability of Rs239.2 million for the current year been charged to revenue. Had the bank continued the earlier policy, profit for the year 2002-03 would have been higher by Rs.239.2 million. F- 78 3. In accordance with principles enunciated in AS-26 “Intangible Assets”, the Bank changed accounting policy in respect of acquisition of software system, the cost of acquisition whereof has been treated as Intangible Assets, to be amortized over the economic life of such assets, maximum up to 5 years. Up to the previous year, such expenses were charged to revenue. Had the bank continued the earlier policy, the profit for the year would have been lower by Rs 371.6 million. VII NOTES ON CONSOLIDATED ACCOUNTS 1. The subsidiaries (which alongwith Punjab National Bank, the Parent, constitute the Group), considered in the preparation of the consolidated financial statements are: Name of the Company Country of % Voting % Voting % Voting incorporation power held power held power held as at as at as at 31st March, 31st March, 31st March, 2002 2003 2004 i) PNB Gilts Ltd. (PNB Gilts) India 74.79 74.79 74.07 ii) PNB Housing Finance Ltd. India 100 100 100 (PNB Housing) iii) PNB Asset Management Co.Ltd. India 100 100 100 (PNB AMC) iv) PNB Capital Services Ltd. (PNB Caps) India 100 100 NA v) PNB Securities Ltd. India 100 NA NA 2. Accounting for investment in following associates has been considered in consolidation based on equity method as provided in Accounting Standard-23 S.No. Name of the Associate Company Country of Proportion of Proportion of Proportion of incorporation ownership ownership ownership percentage percentage percentage 31.03.2002 31.03.2003 31.03.2004 1. Bhojpur Rohtas Grameen Bank . India 35% 35% 35% 2. Magadh Grameen Bank India 35% 35% 35% 3. Nalanda Grameen Bank India 35% 35% 35% 4. Pataliputra Grameen Bank India 35% 35% 35% 5. Haryana Kshetriya Grameen Bank India 35% 35% 35% 6. Hissar Sirsa Kshetriya Grameen Bank. India 35% 35% 35% 7. Ambala Kurukshetra Grameen Bank. India 35% 35% 35% 8. Kapurthala Ferozpur Kshetriya Grameen.Bank India 35% 35% 35% 9. Gurdaspur and Amritsar Kshetriya Grameen Bank India 35% 35% 35% 10. Shekhawati Grameen Bank India 35% 35% 35% 11. Alwar Bharatpur Anchalik Grameen Bank India 35% 35% 35% 12. Kisan Grameen Bank India 35% 35% 35% 13. Devipatan Kshetriya Grameen Bank India 35% 35% 35% 14. Muzaffarnagar Kshetriya Grameen Bank India 35% 35% 35% 15. Hindon Grameen Bank India 35% 35% 35% 16. Vidur Grameen Bank India 35% 35% 35% 17. Himanchal Grameen Bank India 35% 35% 35% 18. Rani Laxmibai Kshetriya Grameen bank India 35% 35% 35% 19. Shivalik Kshetriya Grameen Bank India 35% 35% 35% 20. Everest Bank Ltd. Nepal 20% 20% 20% 21. Asset Care Enterprises Ltd India NA NA 26% F- 79 3. Related party disclosures (AS-18): Name of the related party and their relationship with PNB Group: Key Management Personnel: Punjab National Bank: Shri S.S.Kohli, Chairman & Managing Director Shri T.S.Narayanasami, Executive Director PNB Gilts Ltd. Shri Arun Kaul, Managing Director ( upto May 2003) Shri I.D.Singh, Managing Director (w.e.f. May 2003) PNB Housing Finance Ltd. Shri V.P.Chaudhary, Managing Director (upto 21st Oct.2003) Shri V.K.Sood, Managing Director (w.e.f. 16th Dec.2003) Shri R.Nambirajan (Retired on 31st Aug.2002) Shri Manu Chadha, Director PNB Assets Management Co. Ltd. Shri Ranjan Dhawan, Managing Director (upto 05th July 2003) Shri B.B.Aggarwal, Managing Director (after 05th July 2003) PNB Capital Services Ltd Mrs. Sudeshna Sharma, Managing Director Transactions of Key Management Personnel: Amount (Rs. In million) Item 31.03.2002 31.03.2003 31.03.2004 Remuneration 2.5 2.6 2.4 Intt on Deposits Not significant Not significant Not significant Deposits Not significant - 0.1 Loans and Advances - - 0.3 Sitting Fee, out of pocket exp & Concurrent audit fee 0.6 - - 4) Earning per Share (AS-20): 31.03.2002 31.03.2003 31.03.2004 a) Earning per Share (In Rs.) i) Basic Rs.31.64 Rs.35.79 Rs.45.15 ii) Dilutive* Rs.31.64 Rs.35.79 Rs.45.15 b) Amount used as numerator Profit after tax (Rs in Million) 6,715.0 9,395.7 11,979.8 c) Nominal value of shares Rs.10/- per share Rs.10/- per share Rs.10/- per share d) Weighted average number of equity shares used as the denominator 212,241,300 262,540,410 265,302,500 There are no dilutive potential shares F- 80 5. Pending final clearance/ adjustment, the impact of reconciliation of inter-branch transactions, draft paid without advice, clearing imprest, refund paid/payable, balancing of subsidiary ledgers with control ledgers at some of the branches, could not be ascertained exactly but may not be significant. 6. Pending registration of title deeds, properties amounting to Rs.314.8 million (31st March 2004) are included in Premises. 7. a) In compliance with Accounting Standard-22 on “Accounting for Taxes on Income” issued by Institute of Chartered Accountants Of India, Deferred Tax Assets and Deferred Tax Liabilities have been recognised as per accounting policy of the Bank and its subsidiaries. Major components of which are set out below: (Rs.in Million) Particulars As on As on As on 31st March 2002 31st March 2003 31st March 2004 Deferred Tax Assets Provision for Bad & Doubtful Debts 1303.6 410.9 571.4 Provision for Restructured Accounts 0 392.3 433.1 Statutory Liability 6.3 6.4 13.1 Provision for leave encashment 91.9 480.2 561.6 Others 20.6 19.4 18.6 Total 1422.4 1309.2 1597.8 Deferred Tax Liabilities Depreciation on Fixed Assets 290.9 240.0 220.5 VRS Expenditure 363.3 242.7 119.2 Software Expenditure 1.0 115.9 80.0 Others 5.5 - 0.7 Total 660.7 598.6 420.4 Deferred Tax Assets (Net) 761.7 710.6 1177.4 b) No provision is considered necessary in respect of disputed Income Tax demands of Rs.1503.4 million (31st March 2002), Rs.200.2 million (31st March 2003), Rs.405.2 Million (31st March 2004) as in the Bank’s view, duly supported by expert opinion and decision on bank’s own appeals on same issues, additions/ disallowances made by the Assessing Officer are not sustainable. 8. “Other Assets – Others”, includes expenditure on VRS Rs.1160.8 million as on 31st March 2004 to be amortised in subsequent accounting years. 9. In terms of Govt. of India notification dated 31st January 2003, the erstwhile Nedungadi Bank Ltd. was amalgamated with the Bank and accordingly as on and from the prescribed date i.e. 01st Feb. 2003 the business, properties, assets and Liabilities have been taken over by PNB in terms of scheme of amalgamation. The accounting treatment for the amalgamation is given on the basis of ‘Purchase Method’ in the year 2002-03. The asset classification, income recognition and provisioning norms in respect of advances categorized as ‘Advances considered good and readily realisable’ have been applied on ongoing basis. Depreciation on fixed assets acquired from eNBL is provided for the period, such assets are held by the bank. Excess of liabilities over the Assets amounting to Rs.497.7 million, being cost of business have been considered as Goodwill. 10. The bank has approved the restructuring of the Investment in IFCI, carried in “Available for Sale” category. The same involves rollover of the principal, conversion of the interest not due in bonds including Zero coupon bonds, reduction of coupon rate for preference shares and bonds, effective 01st April 2002. In terms of permission granted by the Reserve Bank of India for the year, the post restructuring exposure to IFCI, has been shifted to “ Held to maturity” category at book value in the year 2002-03. As per policy consistently followed by the bank F- 81 for transfer of securities, the difference between the market value and book value amounting to Rs.1341.9 Million as been provided for, though the bank has obtained permission from Reserve Bank of India that the restructuring and transfer of IFCI bonds to ‘ Held to maturity’ category will not attract the prudential norms. 11. The financial statements of PNB Caps for the year 2002-03 have been prepared on the basis of going concern concept, though the Company has stopped accepting any fresh business following RBI’s advice to the bank to take steps to close the Company. 12. In terms of scheme of amalgamation, approved by the Board of Punjab National Bank in its meeting held on 01st March 2003 and sanctioned by the Hon’ble High Court of Delhi vide order-dated 25th August 2003, PNB Capital Services Ltd, a wholly owned subsidiary was merged with the Parent w.e.f. the appointed date 01st April 2003. The amalgamation has been accounted in the year 2003-04 for under “Pooling of Interest Method” as prescribed in AS-14.The accumulated losses amounting Rs.16.1 Million (net of Reserves) have been debited to Reserves (Revenue & Other Reserves). 13. Financial information for the year ended 31st March 2002 are as regrouped / rearranged / reclassified in the year ended 31st March 2003. F- 82 ANNEXURE I SUMMARY OF ACCOUNTING RATIOS (all values are in terms of Rs. in million except otherwise stated) Year ended 31st March 2000 2001 2002 2003 2004 9/2003 9/2004 (half year) (half year) 1. EARNINGS PER SHARE Profit after tax 4,081.4 4,636.4 5,623.9 7,894.9 10,611.1 5,277.4 7,909.3 Weighted average number of equity shares (no.) * 212,241,300 262,540,410 265,302,500 265,302,500 265,302,500 Earning Per Share (Rs.) 26.5 30.1 40.0 19.9 29.8 $ 2. NET ASSET VALUE Share Capital 2,122.4 2,122.4 2,122.4 2,653.0 2,653.0 2,653.0 2,653.0 Total Reserves and Surplus 20,603.5 24,569.6 30,039.5 37,149.8 46,462.2 42,368.3 54,328.7 Less: Revaluation Reserves 3,552.2 3,466.7 3,381.3 3,295.8 3,210.4 3,253.1 3,167.6 Intangible Assets - - 371.6 339.5 359.3 328.2 381.6 Deferred Tax Assets - - 735.5 986.5 1,704.0 1,365.3 2,469.1 Net Asset Value 19,173.7 23,225.3 27,673.5 35,181.0 43,841.5 40,074.7 50,963.4 3. RETURN ON NETWORTH (%) 21.3 20.0 20.3 22.4 24.2 13.2 15.5 $ * unclassified consolidated $ half yearly figures not annualized As per the guidelines of the Reserve Bank Of India, unamortised expenditure on voluntary retirement scheme is not to be subtracted while computing capital adequacy (Tier I Capital). Similar principle has been applied while calculating net asset value / networth. F- 83 Key financial ratios during period are as under: Year ended 31st March Half years ended*** 2000 2001 2002 2003 2004 Sep-03 Sep-04 Net NPA to Net Advances Ratio (%) 8.52 6.74 5.32 3.86 0.98 2.47 0.30 Interest Income / working Fund (%) 10.12 9.84 9.50 9.27 8.12 4.21 3.69 Non Interest Income / working Fund (%) 1.43 1.31 1.40 1.55 1.95 1.16 0.94 Return on Assets ( %) 0.75 0.73 0.77 0.92 1.04 0.57 0.72 Operating Profit / working funds (%) 1.61 1.59 2.11 2.82 3.30 1.88 1.45 Business per employee (Rs.million) 10.65 14.20 16.80 19.56 22.82 20.37 24.85 Net Profit per employee (Rs million) 0.06 0.08 0.10 0.13 0.18 0.09 0.13 Capital Adequacy Ratio (%) 10.31 10.24 10.70 11.91 12.79 12.35 12.45 Tier I (%) 6.73 6.84 6.34 7.00 6.70 7.65 7.31 Tier II (%) 3.58 3.40 4.36 4.91 6.09 4.70 5.14 OTHER KEY RATIOS Year ended 31st March Half years ended* 2000 2001 2002 2003 2004 Sep-03 Sep-04 Credit/Deposit Ratio (%) (net) 47.54% 49.93% 53.60% 53.06% 53.72% 50.13% 54.84% Interest spread/Average working fund (%) 3.17 3.42 3.28 3.87 3.78 1.91 1.69 Gross profit/Average working fund (%) 1.61 1.59 2.11 2.82 3.30 1.90 1.45 Operating exp./Avg. working funds (%) 2.99 3.14 2.57 2.55 2.47 1.18 1.17 Return on Average net worth (%) 23.49 21.87 22.10 25.12 26.86 14.03 16.69 Yield on advances(%) 11.99 11.12 10.67 10.04 9.08 4.73 4.16 Yield on investments(%) 11.73 11.24 10.93 10.38 9.12 4.75 4.11 Cost of deposits(%) 7.69 7.09 6.94 6.16 5.01 2.64 2.31 Cost of Borrowings (%) 5.94 6.04 8.24 2.37 1.32 0.40 0.93 Gross profit per employee (Rs. million) 0.13 0.16 0.25 0.39 0.54 0.29 0.28 Business per branch (Rs.million) 178.89 213.38 251.66 285.83 333.86 296.85 361.87 Gross profit per branch (Rs. million) 2.13 2.44 3.82 5.65 7.85 4.28 4.05 ** Unaudited and not annualised. F- 84 ANNEXURE J CAPITALISATION STATEMENT Rs in million Particulars As on 31st March 2004 Loan Funds Long Term 28,940.3 Short Term 8,364.4 Total debts 37,304.7 Shareholders’ Fund Share Capital 2,653.0 Reserves & Surplus 41,188.5 Total Equity 43,841.5 Long term debt/Equity Ratio 0.66 Notes: 1. Deposits outstanding as on 31st March 2004 of Rs. 879,163.9 million not considered for computing Loan funds. 2 Reserves and surplus are after excluding revaluation reserve, intangible assets and deferred tax asset. 3. As per the guidelines of the Reserve Bank Of India, unamortised expenditure on voluntary retirement scheme is not to be subtracted while computing capital adequacy (Tier I Capital). Similar principle is applied while while calculating shareholder’s funds. 4. Post issue long term debt to equity ratio cannot be determined pending completion of book building process F- 85 ANNEXURE K STATEMENT OF TAX SHELTERS Amount in million FINANCIAL YEAR ENDED 31ST MARCH Half Years Ended* 2000 2001 2002 2003 2004 2003 SEPT 2004 SEPT TAX RATE (%) 38.5 39.6 35.7 36.8 35.9 35.9 36.6 TAX AT ACTUAL RATE ON PROFIT ADJUSTMENTS 2,080.8 2,272.0 2,715.8 4,209.8 6,479.1 2,984.3 3,726.4 PERMANENT DIFFERENCES I INTEREST ON TAX FREE BONDS (862.6) (927.7) (897.3) (873.7) (836.9) (428.1) (250.6) ii Dividends (EXEMPT FROM TAX) (691.2) (309.9) (527.7) (475.9) (581.7) (267.3) (247.6) iii INTEREST INCOME FROM INFRASTRUCTURE PROJECT (715.4) (1,339.3) (906.6) (755.5) (1,041.2) (300.0) (565.2) iv Others 0.0 6.4 295.7 206.5 187.3 3.0 (1,372.0) TOTAL (A) (2,269.2) (2,570.5) (2,035.9) (1,898.6) (2,272.5) (992.4) (2,435.4) TIMING DIFFERENCE I DIFFERENCE BETWEEN BOOK DEPRECIATION AND TAX DEPRECIATION (38.5) 58.5 (107.0) 121.5 63.0 (45.5) (41.8) II PROVISION FOR BAD AND DOUBTFUL DEBTS/ WRITTEN OFF ACCOUNTS 0.0 (1,070.0) 0.0 1,591.3 974.9 745.6 (156.2) III AMORTISATION OF VRS , LEAVE ENCASHMENT, SOFTWARE EXP 0.0 (1,297.6) 187.4 680.4 661.2 342.4 304.0 IV Goodwill 0.0 0.0 0.0 373.3 (373.3) 70.0 0.0 V Others (55.4) 0.7 11.9 (149.3) 85.3 76.0 2,820.0 TOTAL (B) (93.9) (2,308.4) 92.3 2,617.2 1,411.1 1,188.5 2,926.0 NET ADJUSTMENTS (A+B) (2,363.1) (4,878.9) (1,943.6) 718.6 (861.4) 196.1 490.6 TAX SAVINGS THEREON (909.8) (1,929.6) (693.9) 264.1 (309.0) 70.4 179.5 F- 86 Amount in million FINANCIAL YEAR ENDED 31ST MARCH Half Years Ended* 2000 2001 2002 2003 2004 2003 SEPT 2004 SEPT TOTAL TAXATION - Current Year 1,171.0 342.4 2,022.0 4,473.9 6,170.0 3,054.7 3,905.9 Tax relating to Previous Year 69.0 765.9 128.9 (1,122.5) 892.5 (5.7) Deferred Tax (net) 0.0 0.0 (167.4) 55.2 (462.6) (251.2) (1,069.0) Debited to Provisions & Contingencies as per profit and loss account before restatement 1,240.0 1,108.3 1,983.5 3,406.6 6,599.9 2,803.5 2,831.2 Impact of adjustments (306.2) (254.9) (127.5) 303.9 Adjusted Amount debited to Profit and Loss a/c 1,240.0 1,108.3 1,983.5 3,100.4 6,345.0 2,676.0 3,135.1 * Unaudited F- 87 ANNEXURE L Statement of Borrowings Rs. In Million FINANCIAL YEAR ENDED 31ST MARCH Half Years Ended* 2000 2001 2002 2003 2004 30.9.03 30.9.04 Reserve Bank of India 5,133.5 3,000.0 2,690.0 - 43.4 - - Other Banks 107.7 436.5 319.6 1,842.1 5,724.9 3,546.5 5,942.1 IDBI Refinance 0.2 0.1 - - - - - SIDBI Refinance 380.6 2,163.1 97.5 3,123.9 116.6 116.1 86.7 NABARD Refinance 846.3 1,006.6 928.3 642.9 375.0 491.2 376.3 EXIM Refinance 93.1 54.7 6.4 - - - - Borrowings Outside India 62.9 71.0 43.9 1,012.7 6,630.7 3,298.7 13,115.1 Sub total 6,624.3 6,732.0 4,085.7 6,621.6 12,890.6 7,452.5 19,520.2 VRS Bonds - 2,025.1 946.6 886.8 835.5 859.9 815.9 Tier -II Bonds 8,497.7 8,898.5 11,798.6 15,928.6 23,578.6 18,578.6 23,428.6 TOTAL 15,122.0 17,655.6 16,830.9 23,437.0 37,304.7 26,891.0 43,764.7 Break up for tier II bonds Series I 1,999.2 ------Series II 1,899.9 1,899.9 - - - - - Series III 3,998.6 3,998.6 3,998.6 3,978.6 3,978.6 3,978.6 3,978.6 Series IV 600.0 600.0 600.0 600.0 600.0 600.0 600.0 Series V - 2,400.0 2,400.0 2,400.0 2,400.0 2,400.0 2,400.0 Series VI - - 3,000.0 3,000.0 3,000.0 3,000.0 3,000.0 Series VII - - 1,800.0 1,800.0 1,800.0 1,800.0 1,800.0 Series VIII - - - 950.0 950.0 950.0 950.0 Series VIII - - - 2,950.0 2,950.0 2,950.0 2,950.0 Series IX - - - - 2,650.0 2,650.0 2,650.0 Series X - - - - 5,000.0 - 5,000.0 Series 97 (eNBL) - - - 150.0 150.0 150.0 - Series 97 (eNBL) - - - 76.5 76.5 76.5 76.5 Series 2000 (eNBL) - - - 23.5 23.5 23.5 23.5 8,497.7 8,898.5 11,798.6 15,928.6 23,578.6 18,578.6 23,428.6 Notes *** Unaudited 1. All the above loans are unsecured. This statement excludes deposits(demand and term deposits, savings bank) F- 88 SUMMARY OF SIGNIFICANT DIFFERENCES BETWEEN INDIAN GAAP AND U.S. GAAP The summarized financial information and financial statements included in this Red Herring Prospectus have been prepared in accordance with the requirements of the Companies Act and the Banking Regulation Act and accounting principles generally accepted in India (collectively “Indian GAAP”), which differ in certain respects from the accounting principles generally accepted in the United States (or “U.S. GAAP”). The following table summarizes significant measurement differences between U.S. GAAP and Indian GAAP insofar as they affect financial information reported in this Red Herring Prospectus. Various U.S. GAAP and Indian GAAP pronouncements have been issued for which the mandatory application date is later than the reporting dates in this Red Herring Prospectus. These, together with standards that are in the process of being developed in both jurisdictions, could have a significant impact on future comparisons between U.S. GAAP and Indian GAAP. Particulars Indian GAAP U.S. GAAP Format and content of Entities are required to present balance All entities are required to present balance financial statements sheets, profit and loss accounts and, if listed sheets, income statements, statements of or proposing listing, cash flows for two years shareholders’ equity, cash flows and together with accounting policies, schedules comprehensive income, together with and notes. Entities seeking a listing are accounting policies and notes to the financial required to present five years of adjusted statements. The extent of disclosures in the financial information. notes to financial statements generally is far Format for presentation of financial more extensive than under Indian GAAP. statements is as prescribed by the relevant No specific format is mandated, generally items statute. are presented on the face of the Balance Sheet in decreasing order of liquidity. Income statement items may be presented using a single-step or a multiple step format. Expenditure must be presented by function. Allowance for credit losses Allowance for credit losses are based on Loans are identified as non- performing and defaults expected both on principal and placed on non- accrual basis, where interest. The allowance does not consider management estimates that payment of present value of future inflows. The interest or principal is doubtful of collection. allowances are made in accordance with the Non-performing loans are reported after prudential norms prescribed by the Reserve considering the impact of impairment. The Bank of India (RBI). impairment is measured by comparing the carrying amount of the loan to the present value of expected future cash flows or the fair value of the collateral (discounted at the loan’s effective rate). Investments in securities Securities are classified as held to maturity, Investments in marketable equity and all debt available for sale and held for trading as per securities are classified according to RBI guidelines. Held to maturity are valued management’s holding intent, into one of the at cost unless more than face value in which following categories: trading, available for sale, case the premium is amortized over the or held to maturity. remaining period / years of maturity. Held for trading securities are valued scrip- wise Trading securities are marked to fair value, with and net depreciation is accounted for. the resulting unrealized gain or loss recognized Available for sale are valued scrip- wise and in the income statement. net depreciation under each category is provided for while net appreciation is ignored. 270 Particulars Indian GAAP U.S. GAAP Amortisation of purchase premium is Available-for-sale (AFS) securities are marked required in respect of ‘Held to Maturity’ to fair value, with the resulting unrealized gain category or loss recorded directly in a separate component of equity called ‘Other Comprehensive Income’ until realized, at which time the gain or loss is reported in income. Held-to-maturity (HTM) debt securities are carried at amortized cost. Other than temporary impairments in the value of HTM and AFS investments are accounted for as realized losses. Amortisation of purchase premium and discount is required for all the categories of debt securities. Loan origination fees/costs Loan origination fees and costs are taken to Loan origination fees (net of loan origination the income statement in the year accrued/ costs) are deferred and recognized as an incurred. adjustment to yield over the life of the loan. Consolidation and In India, the reporting entity generally Under U.S. GAAP, there is a presumption that investments in subsidiaries follows legal form, and under the consolidated financial statements present more Companies Act is considered to be the legal meaningful financial information for a parent entity rather than a group. and subsidiaries than separate financial statements of the parent. Accordingly, there is no legal requirement to prepare consolidated financial Accordingly, consolidation is required for statements. In stand alone financial entities where the parent has majority financial statements, investments in subsidiaries, if control, generally when it controls more than classified as long term investments, are 50% of the outstanding voting stock, except accounted at cost less an allowance for when control is likely to be temporary or is permanent impairments. If disclosed as impaired. Separate financial statements of the current investments, they are valued at parent only are not presented. lower of cost and fair value. Entities where the minority shareholder has Accounting Standard (AS21) on substantive participating rights overcome the “Consolidated Financial Statements”, does presumption that the majority shareholder not require consolidation, but sets out the controls the entity thus precluding standards to be followed in the event that consolidation of the results of that entity. In consolidated financial statements are such cases, the equity method of accounting presented or required by law or regulation. applies. SEBI requires listed companies and those Entities where the minority shareholder has seeking a listing to publish consolidated protective rights only are consolidated. financial statements in accordance with AS21 in addition to the separate financial For the purposes of identifying the voting statements of the parent. interests held in an investee, all direct and indirect interests are considered. Accordingly, certain investees may be considered as 271 Particulars Indian GAAP U.S. GAAP For the purposes of identifying the voting subsidiaries to be consolidated under U.S. GAAP, interests held in an investee, direct interests which may be treated as equity affiliates under and those indirect interests held through a Indian GAAP. subsidiary are considered. In January 2003, the FASB issued Interpretation Unlisted entities with subsidiaries will No. 46, “Consolidation of Variable Interest continue to have the option of not presenting Entities” an interpretation of Accounting consolidated financial statements. Research Bulletin (ARB) 51 that applies to variable interest entities created after January 31, 2003, and to variable interest entities in which an enterprise obtains an interest after that date. A variable interest entity to be consolidated is one in which a party could face risk of loss without having an equity interest, and includes many entities that would previously have remained off-balance sheet. Investments in associates or The equity method of accounting for Investments over which the investor can exert affiliates investments in associates is required in significant influence, generally presumed when consolidated financial statements of listed the investor owns between 20% and 50% of companies. The definition of associates and the voting stock, are required to be accounted equity accounting are essentially similar to for using the equity method. US GAAP. There is no requirement to apply The equity method requires investors to record the equity method of accounting in the their investment in the associate as a one-line standalone financial statements of the parent asset and reflect their share of the investee’s and the same are accounted for in the same net income/loss in their earnings. Dividends manner as other investments in the stand received reduce the investment account. alone financial statements of a parent. This method is also followed for unconsolidated Unlisted companies that do not prepare subsidiaries. consolidated financial statements could continue to account joint venture investments as before. Interests in joint ventures Investment in jointly controlled entities is An incorporated joint venture is treated as a accounted in stand alone financial subsidiary or an affiliate, depending on the level statements of the parent in the same manner of control of the joint venturer, and either as stated in ‘Consolidation and investment consolidated or accounted for using the equity in subsidiaries’ above. method, respectively. Interests in jointly controlled entities of a Accounting for interests in jointly-controlled venture should be recognized in its assets and jointly-controlled operations of a consolidated financial statements on a venture is similar to Indian GAAP. proportionate consolidation basis unless by virtue of a contractual arrangement joint control is established over an entity which is a subsidiary of that enterprise within the meaning of AS 21, in which case the entity is consolidated by the said enterprise and is not treated as joint venture. Additionally, 272 Particulars Indian GAAP U.S. GAAP interests in jointly-controlled assets and jointly-controlled operations of a venture are required to be recognized in the separate financial statements and consequently in its consolidated financial statements. Unlisted companies that do not prepare consolidated financial statements could continue to report joint venture investments as before. Business combination Business combinations are accounted for The ‘Purchase method’ of accounting is required either as pooling of interests or as for all business combinations. SFAS No. 141 acquisitions. Accounting for business requires intangible assets to be recognized if combinations as pooling of interests is they arise from contractual or legal rights or are permitted only on fulfillment of certain “separable”, i.e., it is feasible that they may be conditions. Non-fulfillment of one or more sold, transferred, licensed, rented, exchanged conditions results in the combination being or pledged. accounted for as an acquisition using the Under APB Opinion No. 16, the pooling of ‘purchase method’ of accounting. interest method is required in respect of Under the pooling of interest method, the combination of entities under common control assets, liabilities and reserves of the in a manner similar to Indian GAAP. transferor company are recorded by the Under purchase accounting, the consideration transferee company at their existing carrying is measured at fair value, the purchase price amounts after making changes for allocated to the fair values of the next assets uniformity of accounting policies. acquired including intangibles, and goodwill Under the purchase method, assets and recognized for the difference between the liabilities are recorded either at their existing consideration paid and the fair value of the net carrying amounts or by allocating the assets acquired. consideration to individual identifiable assets and liabilities on the basis of their fair values at the date of acquisition. Acquired Goodwill Goodwill arising on amalgamation is Under SFAS No. 142, effective for fiscal years amortised to income on a systematic basis beginning after December 15, 2001, goodwill over its useful life, not exceeding five years arising on new acquisitions and any unamortized unless a longer period can be justified. (AS balance for prior acquisitions will no longer be 14) The amount of goodwill recognized is subject to amortization. Instead, such goodwill the difference between the consideration will be tested for impairment on an annual basis paid and the book value of the net assets or whenever triggers indicating impairment acquired. Negative goodwill is credited to a arise. The impairment test is based on estimates capital reserve. of fair value at a reporting unit level. Goodwill arising on the acquisition of shares of a company is generally not separately recognized, but is included in the cost of the investment. 273 Particulars Indian GAAP U.S. GAAP For companies that prepare consolidated financial statements, goodwill arising on consolidation is recognized upon consolidation. Such goodwill is not amortized. Additionally, goodwill needs to be tested for impairment on annual basis, as required by AS 28 “Impairment of Assets”. Impairment of assets Applicable for accounting periods beginning SFAS No.144 develops one accounting model from April 1, 2004 onwards. for long-lived assets other than goodwill that are to be disposed of by sale, as well as The standard required companies to assess addresses the principal implementation issues. whether there is any indication that an asset is impaired at each balance sheet date. If such SFAS No.144 requires that long-lived assets that an indication exists, the company is required are to be disposed of by sale be measured at to estimate the recoverable amount of the the lower of book value or fair value less cost to asset. If the recoverable amount of an asset sell. is less than its carrying amount, that carrying The impairment review is based on amount of the asset should be reduced to undiscounted cash flows at the lowest level of its recoverable amount. That reduction is independent cash flows. If the undiscounted reported as an impairment loss. cash flows are less than the carrying amount, the impairment loss must be measured using discounted cash flows. Property, plant and Fixed assets are recorded at historical costs Revaluations are not permitted. equipment or revalued amounts. On revaluation, an entire class of assets is revalued, or a selection of assets for revaluation is made on a systematic basis. There is no restriction on the frequency of revaluation. However, revaluation should not exceed the recoverable amount of assets. Issuance and redemption Debt issuance costs may be amortized, Debt issuance costs are treated as a deferred costs for borrowings charged as an expense or charged to the charge and amortized using the effective Securities Premium Account. interest rate method over the life of the debt. Redemption premiums are accrued as a yield Redemption premiums payable on the adjustment over the life of the debt. redemption of debt may be accrued over the life of the debt. Foreign exchange AS11 “The Effects of Changes in Foreign Under US GAAP gains or losses arising from Exchange Rates” deals with accounting for foreign currency transactions are included in foreign exchange transactions. Transactions determining net income. Foreign exchange in foreign currency are recorded at the gains or losses are not included in interest cost. 274 Particulars Indian GAAP U.S. GAAP exchange rate prevailing on the date of the For the purposes of consolidating a foreign transaction. Monetary items are restated at subsidiary, its financial statements are year-end exchange rates. Exchange translated into the parent’s reporting currency. differences arising on transactions and Assets and liabilities are translated using the translation of monetary items are recognized balance sheet rate of exchange. Amounts in as income or expense in the year in which the income statements are translated using the they arise. Foreign exchange losses that weighted average rate for the period. relate to foreign borrowings incurred to Translation differences that arise are reported finance an asset are treated as a part of in a separate component of shareholders’ borrowing cost and are capitalised. equity. With the revision of this standard, with effect from accounting periods commencing on or after April 1, 2004, translation differences arising on a monetary item that, in substance, forms part of an enterprise’s net investment in a non-integral foreign operation should be accumulated in a foreign currency translation reserve in the enterprise’s financial statements until the disposal of the net investment, at which time they should be recognized as income or as expenses. Guidance relating translation of foreign operations integral to the reporting enterprise requires foreign exchange gains or losses to be recognized in the income statement. Deferred taxation Deferred taxes are required to be provided Deferred tax liabilities and assets are recorded for the tax effect of timing differences for the tax effect of temporary differences between taxable income and accounting between the tax and book bases of assets and income using substantively enacted tax liabilities and operating loss carry-forwards, at rates. currently enacted tax rates expected to be in force when the temporary differences reverse. Deferred tax assets arising due to Changes in tax rates are reported in the income unabsorbed depreciation or carry forward of statement in the period of enactment. losses are recognized only to the extent that there is virtual certainty that sufficient future A valuation allowance is made against deferred taxable income will be available against taxes if, based on the weight of available which such deferred tax assets can be evidence, it is more likely than not that some realized. portion or all of the deferred tax asset will not be realized. Other deferred tax assets are recognized and carried forward only to the extent that there is a reasonable certainty that sufficient future taxable income will be available against which such deferred tax assets can be realized. 275 Particulars Indian GAAP U.S. GAAP Proposed dividend Proposed dividends are recognised in the Dividends are recorded in the year of financial statements in the period to which declaration. they relate, even if they are subject to shareholders’ approval. Vacation accrual Vacation accrual, or leave encashment, is Vacation earned but not taken is reported as a viewed as a retirement entitlement and is liability based on the number of days generally reported at the actuarially entitlement, priced at the balance sheet salary determined present value of future benefits. rate. Retirement benefits The liability for defined benefit retirement The liability for defined benefit retirement plans plans is reported at an actuarial valuation. is reported at the present value of future Several alternative methodologies are benefits using the projected unit credit method, considered acceptable for the purposes of with a stipulated method to determine the valuation, and the actuary has assumptions. considerable latitude in selecting Expenditure incurred on voluntary retirement assumptions to be used. scheme should be expensed in the period Expenditure incurred on voluntary incurred. retirement scheme may be deferred. Depreciation Depreciation is generally charged at rates Depreciation is provided in a systematic and prescribed by the Companies Act. These rational manner over the estimated useful rates are the minimum rates, and companies economic life of the assets. are permitted to charge depreciation at higher rates, in order to write off the cost of assets over their useful lives, if shorter. Derivative financial The Guidance note on Accounting for Equity All derivatives are required to be recognised as instruments and hedging Index Options and Equity Stock Options and assets or liabilities in the balance sheet and the guidance note on accounting for equity measured at fair value. The accounting for index futures are the pronouncements, which changes in the fair value of a derivative (that is address the accounting for derivatives. gain and losses) depends on the intended use of the derivative and the resulting designation. RBI Guidelines on Forward Rate Agreements Derivatives based on the intended use are / Interest Rate Swaps requires the broadly classified into three classes’ viz. Fair transactions to be classified in to hedging value hedge, Cash flow hedge and Foreign and trading. The swap that is accounted for currency hedge. like a hedge should be accounted for on accrual basis except the swap designated Gains and losses on fair value hedges, for both with an asset or liability that is carried at the hedging instrument and the item being market value or lower of cost or market value hedged, are recognised in the income in the financial statements. In that case the statement. swap should be marked to market with the Gain and losses on effective portion of cash resulting gain or loss recorded as an flow hedges is initially reported as a component adjustment to the market value of designated of other comprehensive and subsequently asset or liability. Gains or losses on the reclassified in to earnings when the forecasted termination of swaps should be recognised transaction affects earnings. when the offsetting gain or loss is recognised on the designated asset or liability. This 276 Particulars Indian GAAP U.S. GAAP implies that any gain or loss on the In case of hedging the foreign currency terminated swap would be deferred and exposure of a net investment in a foreign recognised over the shorter of the remaining operation, the same accounting treatment is contractual life of the swap or the remaining given as in the case of Cash flow hedge. life of the asset/ liability. The gains or losses on the ineffective portion Trading swaps should be marked to market of any hedge are written off as income or with changes recorded in the income expense. statement. Income and expenses relating Derivatives that are not designated as a hedging to these swaps should be recognised on the instrument, the gain or losses is recognised in settlement date. Gains or losses on the earnings in the period of change. termination of trading swaps should be recorded as immediate expense or income. Off-balance sheet items There is no specific guidance or the SEC registrants are required to provide accounting and reporting for of-balance extensive disclosures of material off-balance sheet items. Commitments and sheet items, contingent liabilities and financial contingencies are required to be disclosed. guarantees. Commitments and contingencies are required to be disclosed. Fair values of financial There is no requirement to disclose the fair Extensive disclosures are required of the fair instruments value of financial instruments. values of financial instruments and the methodologies or determining fair values. 277 MATERIAL CONTRACTS AND DOCUMENTS FOR INSPECTION The following contracts (not being contracts entered into in the ordinary course of business carried on by our Bank or entered into more than two years before the date of this Red Herring Prospectus) which are or may be deemed material have been entered or to be entered into by our Bank. These contracts, copies of which have been attached to the copy of this Red Herring Prospectus, delivered to the Designated Stock Exchange for registration and also the documents for inspection referred to hereunder, may be inspected at the office of our Bank situated at 5, Sansad Marg, New Delhi 110 001 between 10.00 am to 4.00 pm on working days from the date of this Red Herring Prospectus until the Bid Closing Date/Issue Closing Date. Material Contracts 1. Letters of appointment dated October 26, 2004 to ICICI Securities Limited, DSP Merrill Lynch Limited, Enam Financial Consultants Private Limited, JM Morgan Stanley Private Limited and Kotak Mahindra Capital Company Limited from our Bank appointing them as BRLMs. 2. Letters of appointment dated October 25, 2004 to the Registrar to the Issue. 3. Memorandum of Understanding amongst our Bank and the BRLMs dated January 13, 2005. 4. Memorandum of Understanding executed by our Bank and the Registrar to the Issue dated January 28, 2005. 5. Escrow Agreement dated [ ] , 2005 between the Bank, the BRLMs, Escrow Collection Bank and the Registrar to the Issue. 6. Syndicate Agreement dated [ ] , 2005 between the Bank, the BRLMs and the Syndicate Members. 7. Underwriting Agreement dated [ ] , 2005 between the Bank, the BRLMs and the Syndicate Members. Material Documents 1. Opinion from Mr. R. Ganeshan, Chartered Accountant, dated February 5, 2005. 2. Opinion from Mr. Kanwaljit Singh, Chartered Accountant, dated February 2, 2005. 3. Letter dated February 7, 2005 from Punjab National Bank to the Ministry of Finance, Government of India seeking certain clarifications with regard to the Return of Proceeds and the Capital Reduction. 4. Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970 as amended from time to time. 5. Punjab National Bank (Shares & Meetings) Regulations, 2000 as amended from time to time. 6. Letter number F.No.11/29/2004-BOA dated January 7, 2005 from the Banking Division, Department of Economic Affairs, MoF, GoI whereby GoI has given permission for the Issue under Section 3(2B)(c) of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970. 7. Letter number FE.CO.FID/4998/10.02.40(8435)/2004-05 dated February 2, 2005 from the Foreign Exchange Department, RBI permitting us to issue shares to NRIs / FIIs with repatriation benefits out of our public Issue of 80,000,000 Equity Shares. 8. Shareholders’ resolution dated October 11, 2004 in relation to this Issue and other related matters. 9. Resolutions of the Board of Directors dated September 11, 2004 and January 13, 2005 in relation to this Issue and other related matters. 10. Reports of the statutory Auditors dated January 29, 2005 prepared as per Indian GAAP and mentioned in this Red Herring Prospectus. 11. Copies of annual reports of our Bank and our subsidiaries for the last five financial years. 12. Consents of the Auditors being (1) M/s. Surendar K Jain & Co (2) M/s. Mookherjee Biswas & Pathak (3) M/s M.C. Bhandari & Co, (4) M/s P.K.Chopra & Co (5) M/s. Ramanlal G Shah & Co, and (6) M/s. B.K.Ramadhyani & Co for inclusion of their report on accounts in the form and context in which they appear in this Red Herring Prospectus. 13. General Power of Attorney executed by the Directors of our Bank in favour of Person(s) for signing and making necessary changes to this Red Herring Prospectus and other related documents. 14. Consents of BRLMs, Syndicate Members, Registrar to the Issue, Escrow Collection Bank(s), Banker to the Issue, Domestic Legal Counsel to the Bank, International Legal Counsel to the Bank, Directors, Company Secretary and Compliance Officer, as referred to, in their respective capacities. 278 15. Listing applications dated January 17, 2005 filed with NSE and BSE. 16. In-principle listing approvals dated January 18, 2005 and January 28, 2005 from NSE and BSE respectively. 17. Tripartite agreement between NSDL, our Bank and the Registrar to the Issue dated November 13, 2001 in respect of debt instruments and the cover letter extending the same to cover equity shares dated February 26, 2002 bearing reference number AS/PM/02/393 18. Tripartite agreement between CDSL, our Bank and the Registrar to the Issue dated February 25, 2002. 19. Due diligence certificate dated January 14, 2005 to SEBI from ICICI Securities Limited, DSP Merrill Lynch Limited, Enam Financial Consultants Private Limited, JM Morgan Stanley Private Limited and Kotak Mahindra Capital Company Limited. 20. SEBI observation letter no. CFD/DIL/UR/3290/2005 dated February 4, 2005 and our reply to the same dated February 9, 2005. Any of the contracts or documents mentioned in this Red Herring Prospectus may be amended or modified at any time if so required in the interest of the Bank or if required by the other parties, without reference to the shareholders subject to compliance with the applicable laws. 279 DECLARATION All the relevant provisions of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970, Punjab National Bank (Shares & Meetings) Regulations, 2000, Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970 and the guidelines issued by the Government of India or the guidelines issued by Securities and Exchange Board of India, established under Section 3 of the Securities and Exchange Board of India Act, 1992, as the case may be, have been complied with and no statement made in this Red Herring Prospectus is contrary to the provisions of the Banking Companies (Acquisition and Transfer of Undertakings) Act, 1970, Punjab National Bank (Shares & Meetings) Regulations, 2000, Nationalised Banks (Management and Miscellaneous Provisions) Scheme, 1970, the Securities and Exchange Board of India Act, 1992 or rules made thereunder or guidelines issued, as the case may be. We further certify that all statements in this Red Herring Prospectus are true and fair. SIGNED BY ALL DIRECTORS Mr. S.S. Kohli Dr. K.C. Chakrabarty Dr. K.V. Rajan* Mr. P. K. Nayar* Dr. Pritam Singh* Mr. Mohanjit Singh* Mr. A.S. Agarwal* Mr. Mohan Lal Bagga* Dr. K. B. L. Mathur** * Through their constituted attorney Mr. C.P Swarnkar, General Manager, through the respective powers of attorney dated October 18, 2004. ** Through his constituted attorney Mr. C.P Swarnkar, General Manager, through the power of attorney dated January 28, 2005. SIGNED BY MR. ARUN KAUL GENERAL MANAGER, FINANCE Date: February 14, 2005 Place: New Delhi. 280 This page has been left blank intentionally 281 This page has been left blank intentionally 282	https://docslib.org/doc/8570115/punjab-national-bank-constituted-under-the-banking-companies-acquisition-and-transfer-of-undertakings-act-1970-as-punjab-national-bank-on-july-19-1969
16. Diabetes Advocacy: Standards of Medical Care in Diabetes—2019 \| Diabetes Care \| American Diabetes Association The American Diabetes Association (ADA) “Standards of Medical Care in Diabetes” includes the ADA’s current clinical practice recommendations and is intended to 16. Diabetes Advocacy: Standards of Medical Care in Diabetes—2019 American Diabetes Association Diabetes Care 2020;43(Supplement_1):S203–S204 https://doi.org/10.2337/dc20-S016 PubMed: 31862759 The American Diabetes Association (ADA) “Standards of Medical Care in Diabetes” includes the ADA’s current clinical practice recommendations and is intended to provide the components of diabetes care, general treatment goals and guidelines, and tools to evaluate quality of care. Members of the ADA Professional Practice Committee, a multidisciplinary expert committee (https://doi.org/10.2337/dc20-SPPC), are responsible for updating the Standards of Care annually, or more frequently as warranted. For a detailed description of ADA standards, statements, and reports, as well as the evidence-grading system for ADA’s clinical practice recommendations, please refer to the Standards of Care Introduction (https://doi.org/10.2337/dc20-SINT). Readers who wish to comment on the Standards of Care are invited to do so atprofessional.diabetes.org/SOC. Managing the daily health demands of diabetes can be challenging. People living with diabetes should not have to face discrimination due to diabetes. By advocating for the rights of those with diabetes at all levels, ADA can help to ensure that they live a healthy and productive life. A strategic goal of the ADA is for more children and adults with diabetes to live free from the burden of discrimination. The ADA is also focused on making sure cost is not a barrier to successful diabetes management. One tactic for achieving these goals has been to implement the ADA Standards of Care through advocacy-oriented position statements. The ADA publishes evidence-based, peer-reviewed statements on topics such as diabetes and employment, diabetes and driving, insulin access and affordability, and diabetes management in certain settings such as schools, childcare programs, and correctional institutions. In addition to the ADA’s clinical documents, these advocacy statements are important tools in educating schools, employers, licensing agencies, policy makers, and others about the intersection of diabetes medicine and the law and for providing scientifically supported policy recommendations. ADVOCACY STATEMENTS The following is a partial list of advocacy statements ordered by publication date, with the most recent statement appearing first. Insulin Access and Affordability The ADA’s Insulin Access and Affordability Working Group compiled public information and convened a series of meetings with stakeholders throughout the insulin supply chain to learn how each entity affects the cost of insulin for the consumer. Their conclusions and recommendations are published in the following ADA statement. Cefalu WT, Dawes DE, Gavlak G, et al.; Insulin Access and Affordability Working Group. Insulin Access and Affordability Working Group: conclusions and recommendations. Diabetes Care 2018;41:1299–1311 [published correction appears in Diabetes Care 2018;41:1831];https://doi.org/10.2337/dci18-0019(first publication 2018) Diabetes Care in the School Setting A sizable portion of a child’s day is spent in school, so close communication with and cooperation of school personnel are essential to optimize diabetes management, safety, and academic opportunities. See the following ADA position statement for diabetes management information for students with diabetes in the elementary and secondary school settings. Jackson CC, Albanese-O’Neill A, Butler KL, et al.; American Diabetes Association. Diabetes care in the school setting: a position statement of the American Diabetes Association. Diabetes Care 2015;38:1958–1963;https://doi.org/10.2337/dc15-1418(first publication 1998; latest revision 2015) Care of Young Children With Diabetes in the ChildCare Setting Very young children with diabetes have legal protections and can be safely cared for by childcare providers with appropriate training, access to resources, and a system of communication with parents and the child’s diabetes provider. See the following ADA position statement for information on young children aged <6 years in settings such as day care centers, preschools, camps, and other programs. Siminerio LM, Albanese-O’Neill A, Chiang JL, et al.; American Diabetes Association. Care of young children with diabetes in the childcare setting: a position statement of the American Diabetes Association. Diabetes Care 2014;37:2834–2842;https://doi.org/10.2337/dc14-1676(first publication 2014) Diabetes and Driving People with diabetes who wish to operate motor vehicles are subject to a great variety of licensing requirements applied by both state and federal jurisdictions. For an overview of existing licensing rules for people with diabetes, factors that impact driving for this population, and general guidelines for assessing driver fitness and determining appropriate licensing restrictions, see the following ADA position statement. Editor’s note: Federal commercial driving rules for individuals with insulin-related diabetes changed on 19 November 2018. These changes will be reflected in a future updated ADA statement. Lorber D, Anderson J, Arent S, et al.; American Diabetes Association. Diabetes and driving. Diabetes Care 2014;37(Suppl. 1):S97–S103;https://doi.org/10.2337/dc14-S097(first publication 2012) Diabetes and Employment Any person with diabetes, whether insulin treated or noninsulin treated, should be eligible for any employment for which he or she is otherwise qualified. Employment decisions should never be based on generalizations or stereotypes regarding the effects of diabetes. For a general set of guidelines for evaluating individuals with diabetes for employment, including how an assessment should be performed and what changes (accommodations) in the workplace may be needed for an individual with diabetes, see the following ADA position statement. Anderson JE, Greene MA, Griffin JW Jr, et al.; American Diabetes Association. Diabetes and employment. Diabetes Care 2014;37(Suppl. 1):S112–S117;https://doi.org/10.2337/dc14-S112(first publication 1984; latest revision 2009) Diabetes Care in Correctional Institutions People with diabetes in correctional facilities should receive care that meets national standards. Correctional institutions should have written policies and procedures for the management of diabetes and for the training of medical and correctional staff in diabetes care practices. For a general set of guidelines for diabetes care in correction institutions, see the following ADA position statement. American Diabetes Association. Diabetes management in correctional institutions. Diabetes Care 2014;37(Suppl. 1):S104–S111;https://doi.org/10.2337/dc14-S104(first publication 1989; latest revision 2008) Suggested citation: American Diabetes Association. 16. Diabetes advocacy: Standards of Medical Care in Diabetes—2020. Diabetes Care 2020;43(Suppl. 1):S203–S204	https://diabetesjournals.org/care/article/43/Supplement_1/S203/30719/16-Diabetes-Advocacy-Standards-of-Medical-Care-in
Single-dose rifampicin and BCG to prevent leprosy - International Journal of Infectious Diseases The MALTALEP trial compared the efficacy of bacillus Calmette–Guérin (BCG) vaccination followed by single-dose rifampicin (SDR) with BCG vaccination alone in preventing leprosy in household contacts and next-door neighbours of newly diagnosed leprosy patients in Bangladesh ( Richardus et al., 2019 Richardus R. Alam K. Kundu K. Chandra Roy J. Zafar T. Chowdhury A.S. et al. Effectiveness of single-dose rifampicin after BCG vaccination to prevent leprosy in close contacts of patients with newly diagnosed leprosy: a cluster randomized controlled trial. Int J Infect Dis.2019;88:65-72 Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). It was a large, well designed cluster randomised controlled trial (RCT) and 14,988 contacts of 1552 new leprosy patients were randomised to receive either BCG alone (7378) or BCG followed by SDR (7609) 8–12 weeks later. Participants were followed for 2 years. The primary outcome of the RCT recorded in the Netherlands Trial Register, 2019 Netherlands Trial Register. Available from:https://www.trialregister.nl/trial/2940. [Accessed 21 October 2019]. Google Scholar(NTR3087) and reported in the peer-reviewed protocol was “New cases of leprosy among the contacts of index cases” and “…the number of new leprosy patients emerging from the contact groups” respectively ( Richardus et al., 2013 Richardus R.A. Alam K. Pahan D. Feenstra S.G. Geluk A. Richardus J.H. The combined effect of chemoprophylaxis with single dose rifampicin and immunoprophylaxis with BCG to prevent leprosy in contacts of newly diagnosed leprosy cases: a cluster randomized controlled trial (MALTALEP study). BMC Infect Dis.2013;13:456 Crossref PubMed Scopus (41) Google Scholar). There were no significant differences between the proportions of contacts who developed leprosy following the interventions at one or two years. This is an important negative finding and in keeping with the findings of the COLEP RCT of SDR ( Moet et al., 2008 Moet F.J. Pahan D. Oskam L. Richardus J.H. Effectiveness of single dose rifampicin in preventing leprosy in close contacts of patients with newly diagnosed leprosy: cluster randomised controlled trial. BMJ.2008;336:761-764 Crossref PubMed Scopus (163) Google Scholar). The COLEP trial identified that the short-term benefits of SDR were only significant in more distant contacts of index cases. A secondary data analysis was planned to “define special groups at risk for developing leprosy” and “No significant differences of interest were found” ( Richardus et al., 2013 Richardus R.A. Alam K. Pahan D. Feenstra S.G. Geluk A. Richardus J.H. The combined effect of chemoprophylaxis with single dose rifampicin and immunoprophylaxis with BCG to prevent leprosy in contacts of newly diagnosed leprosy cases: a cluster randomized controlled trial (MALTALEP study). BMC Infect Dis.2013;13:456 Crossref PubMed Scopus (41) Google Scholar). The authors discuss the non-significant 42% reduction in the number of new cases of paucibacillary leprosy in those who received SDR after one year. We were surprised that the significant number of individuals who developed multibacillary (MB) leprosy in the SDR group by 2 years was not similarly discussed. The odds of having developed MB leprosy at the two year follow up point were 3.68 (95% CI: 1.03–13.21) in the group randomised to receive BCG and SDR compared to BCG alone. Table 6 states that only one of 11 new cases of MB leprosy diagnosed after completion of the intervention was slit-skin smear positive. The clinical relevance of the increased numbers of MB patients should be discussed including information about nerve function impairment. The COLEP trial did not show any significant difference in the amount of MB disease between the SDR and placebo groups during two or four years of follow up ( Moet et al., 2008 Moet F.J. Pahan D. Oskam L. Richardus J.H. Effectiveness of single dose rifampicin in preventing leprosy in close contacts of patients with newly diagnosed leprosy: cluster randomised controlled trial. BMJ.2008;336:761-764 Crossref PubMed Scopus (163) Google Scholar). The MALTALEP study shows that SDR after BCG does not have a significant protective effect against leprosy in household and next-door neighbour contacts compared to BCG alone. This replicates the finding from the larger COLEP study. Data from the BCG arm of the current study were compared with the placebo arm of the COLEP study to infer that the protective effect of BCG is doubtful in Bangladesh. We agree with Richardus et al. that the current evidence does not support the use of BCG followed by SDR for the contacts of leprosy patients ( Richardus et al., 2019 Richardus R. Alam K. Kundu K. Chandra Roy J. Zafar T. Chowdhury A.S. et al. Effectiveness of single-dose rifampicin after BCG vaccination to prevent leprosy in close contacts of patients with newly diagnosed leprosy: a cluster randomized controlled trial. Int J Infect Dis.2019;88:65-72 Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and with WHO that the evidence for BCG alone is conflicting with no evidence of benefit ( Anon, 2018a BCG vaccines: WHO position paper — February 2018. Wkly Epidemiol Rec.2018;93:73-96 PubMed Google Scholar). The incomplete understanding of the transmission ofMycobacterium lepraeand development of disease hamper the discovery of an effective strategy for prevention. The administration of SDR to contacts of individuals diagnosed with leprosy is recommended by WHO without providing criteria to determine who is a contact ( Anon, 2018b Guidelines for the diagnosis, treatment and prevention of leprosy. World Health Organization, Regional Office for South-East Asia,New Delhi2018 Google Scholar). The evidence for SDR as a strategy to prevent leprosy or achieve the target of zero transmission ofM. lepraeremains limited.	https://www.ijidonline.com/article/S1201-9712(20)30057-6/fulltext
DOL continues misclassification push, says “most workers are employees,” not contractors - Lexology When is a worker really in business for him or herself? This is the fundamental question addressed by the U.S. Department of Labor's (DOL)… When is a worker really in business for him or herself? This is the fundamental question addressed by the U.S. Department of Labor’s (DOL) Administrator’s Interpretation No. 2015-1, released July 15, 2015 (the Interpretation). According to the Interpretation, the answer is “not often.” The Interpretation is a comprehensive reflection of DOL’s view that independent contractor misclassification is common, and it exemplifies DOL’s persistent enforcement approach to this issue. The Fair Labor Standards Act’s (FLSA) definition of “employ” includes “to suffer or permit to work.” 29 U.S.C. § 203(g). Citing Supreme Court precedent, the Interpretation takes a very broad view of when a worker is “suffered or permitted” to work. It notes that the phrase comes from pre-FLSA child labor laws, where its purpose was to prevent employers from disclaiming control over workers as a means of evading child welfare laws. That purpose is still evident in the present day “economic realities” test, applied by the DOL and courts to determine whether a worker is classified correctly under the FLSA. Like pre-FLSA laws, the economic realities test considers multiple factors in addition to the control factor. In the Interpretation’s view, the economic realities test is meant to determine whether the worker is “economically dependent” on the employer on the one hand, or in business for him or herself, on the other hand. To illustrate, the Interpretation analyzes each factor of the economic realities test, asking and answering a familiar set of questions: Is the work an integral part of the employer’s business? Work may be considered integral if it produces the product the company is in the business of selling, and according to the Interpretation, workers who perform such work are more likely to be employees. For example, baseball players are integral to the business of a baseball club, but concessions workers may not be. Does the worker’s managerial skill affect the worker’s opportunity for profit or loss? Per the Interpretation, contractors have the ability to profit by making business decisions. Deciding to work more hours may earn a worker more money, but if the increase is not the result of the worker’s managerial skill, DOL would find this factor weighs towards employee status. How does the worker’s relative investment compare to the employer’s investment? The DOL believes that a contractor should invest in his own business to a degree that is beyond what is necessary to perform the job for the employer. Further, DOL says the investment should not be “relatively minor” compared to the employer’s investment. Does the work performed require special skill and initiative? Many workers have a high degree of technical skills, but the DOL states that a contractor applies those skills in a way that shows business-like initiative, such as by marketing them. Is the relationship between the worker and the employer permanent or indefinite? The Interpretation states that this factor weighs towards contractor status if, as a result of business initiative, a worker works for multiple entities on a project by project basis. On the other hand, permanent or indefinite relationships indicate employee status. What is the nature and degree of the employer’s control? The Interpretation states that contractors exercise actual control over meaningful aspects of the work, and not solely their work schedules. The DOL warns against giving this factor undue weight because it believes economic dependence can exist even when the employer exercises relatively little control. According to the Interpretation, each question should be answered with an eye towards the ultimate determination of whether the worker is an economically independent businessperson. Furthermore, the DOL clearly expects that, in most cases, applying the Interpretation will show that the worker is an “economically dependent” employee. Although the Interpretation is not binding legal authority, it is a clear effort by DOL to advertise its position on worker classification and its intent to conduct enforcement actions accordingly. Notably, the Interpretation does not address classification within the growing “on-demand” economy, at least not outside the frame of the economic realities test. As courts and observers have recognized, the economic realities test does not apply neatly to so-called “dependent contractors,” i.e., those who retain a high degree of economic independence, yet are often dependent on a single employer. See Cotter v. Lyft, Inc., 60 F. Supp. 3d 1067, 1069 (N.D. Cal. 2015) (“At first glance, Lyft drivers don’t seem much like employees . . . [b]ut Lyft drivers don’t seem much like independent contractors either); O’Connor v. Uber Technologies, Inc., No. C-13-3826 EMC, 2015 WL 1069092, at *15 (N.D. Cal. Mar. 11, 2015) (“It is conceivable that the legislature would enact rules particular to the new so-called “sharing economy.”) It remains to be seen whether cases like these will lead to new guidance, or an altogether new designation of workers. In the meantime, DOL continues its enforcement efforts per the guidelines set forth in the Interpretation. In fact, on the same day the Interpretation was released, became the twenty-third stateto sign a joint enforcement agreement, under which DOL and state agencies share information and coordinate enforcement. These developments are a reminder to employers that government agencies view misclassification as a large problem that hinders the broader effort to cover more employees under the FLSA’s minimum wage and overtime requirements. We previously wrote about that effort in the context of DOL’s new proposed overtime rules, which narrow certain overtime exemptions. In light of the Interpretation, employers should proactively review worker classification practices for compliance. Any review should recognize that all working relationships are unique, and must account for all factors of the economic realities test and applicable state law. Locke Lord LLP - Sean Kilian Filed under USA Employment & Labor Litigation Locke Lord LLP Organisations Lyft	https://www.lexology.com/library/detail.aspx?g=379eba56-b939-4aa8-92e9-2a004491177a
Balloon-Occluded Retrograde Transvenous Obliteration for the Treatment of Gastric Varices: Polidocanol Foam Versus Liquid Ethanolamine Oleate \| Request PDF Request PDF \| Balloon-Occluded Retrograde Transvenous Obliteration for the Treatment of Gastric Varices: Polidocanol Foam Versus Liquid Ethanolamine Oleate \| The purpose of this study was to evaluate the clinical results of the management of gastric varices by balloon-occluded retrograde transvenous... \| Find, read and cite all the research you need on ResearchGate Article Balloon-Occluded Retrograde Transvenous Obliteration for the Treatment of Gastric Varices: Polidocanol Foam Versus Liquid Ethanolamine Oleate September 2015 American Journal of Roentgenology 205(3):659-666 DOI: 10.2214/AJR.14.13389 Authors: Chihiro Itou Tokai University Kazunori Myojin Show all 7 authors Hide Abstract The purpose of this study was to evaluate the clinical results of the management of gastric varices by balloon-occluded retrograde transvenous obliteration with polidocanol foam versus ethanolamine oleate. Twenty patients treated with ethanolamine oleate and 21 patients treated with polidocanol foam were enrolled in this study. Early therapeutic effects were assessed mainly by dynamic contrast-enhanced CT. Subjective symptoms, objective findings associated with the procedures, and changes in laboratory data during the obliteration process were evaluated. Rebleeding from gastric varices was assessed after the procedures. Complete obliteration was confirmed in all but one case of early recanalization after treatment with polidocanol foam. One patient died of acute respiratory distress syndrome after treatment with ethanolamine oleate. The total sclerosant volume was significantly lower for 3% polidocanol foam (13.5 ± 6.8 mL) than for 5% ethanolamine oleate (30.6 ± 15.6 mL) (p < 0.01). Polidocanol foam caused fewer severe reactions, including pain, during and after injection. High body temperature, hemoglobinuria, and reactive pleural effusion were not observed with polidocanol foam. The variance in laboratory data values associated with hemolysis was significantly greater with ethanolamine oleate. No postprocedural rebleeding from the gastric varices was observed during a median follow-up time of 39.5 months after procedures with ethanolamine oleate and 34 months after procedures with polidocanol foam. Polidocanol foam can achieve obliteration of gastric varices comparable to that of ethanolamine oleate but with a significantly lower sclerosant dose and reduced risk of hemolysis-induced complications and harmful reactions, including pain and fever. ... Furthermore, the majority of authors conclude that if the Child-Pugh score improves initially in the first 6 to 9 months after BRTO, it then returns to pre-BRTO baseline levels [26,36]. Overall, the technical success rates of BRTO for patients with gastrorenal/splenorenal shunts and gastric varices range from 79% to 100% [18,26,27,35,[41][42][43][44] [45] [46][47][48][49][50][51][52]. In most studies, gastric variceal rebleed rates of patients who had undergone a successful BRTO procedure range between 0% and 20% [18,24,[26][27][28][35][36][37][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. ... ... Overall, the technical success rates of BRTO for patients with gastrorenal/splenorenal shunts and gastric varices range from 79% to 100% [18,26,27,35,[41][42][43][44][45][46][47][48][49][50][51][52]. In most studies, gastric variceal rebleed rates of patients who had undergone a successful BRTO procedure range between 0% and 20% [18,24,[26][27][28][35][36][37][41][42][43][44] [45] [46][47][48][49][50][51][52][53][54][55][56][57][58][59]. In a recent meta-analysis [60] that included 1,016 patients from 24 studies, the technical success rate was found to be 96.4%. ... ACR Appropriateness Criteria® Radiologic Management of Gastric Varices Article J Am Coll Radiol Charles Y. Kim Jason W. Pinchot Osmanuddin Ahmed Eric J Hohenwalter Hemorrhage, resulting from gastric varies, can be challenging to treat, given the various precipitating etiologies. A wide variety of treatment options exist for managing the diverse range of the underlying disease processes. While cirrhosis is the most common cause for gastric variceal bleeding, occlusion of the portal or splenic vein in noncirrhotic states results in a markedly different treatment paradigm. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment. ... BRTO involves occlusion of outflow vein in the portosystemic shunt such as a gastrorenal shunt, which is conducted by applying an occlusion balloon, followed by a sclerosing agent injected directly into the IGVs. [18][19][20][21][22][23][24][25][26][27][28] In this process, several types of sclerosants such as EO, sodium tetradecyl sulfate (STS) [29][30] , and polidocanol are used [31][32] [33] (Table 1). Recently, various modified BRTO techniques had been developed, including vascular plug-assisted retrograde transvenous obliteration (PARTO) [34][35][36] and coil-assisted retrograde transvenous obliteration (CARTO) 37 (Table 2). ... ... Ito et al. reported the retrospective comparisons of BRTO using polidocanol foam (3% polidocanol and air mixture with 1:4 ratio, n=21) and 5% EO (n= 20). 33 The median follow-up periods were 39.5 months (mean 59.2 ± 54.4 months; range 1-135 months) for the EO group and 34 months (mean 31.1 ± 25 months, range 1-97 months) for the polidocanol foam group. Complete obliteration was confirmed in all but one case of early recanalization after treating with polidocanol foam. ... Recent Development of Balloon-Occluded Retrograde Transvenous Obliteration Aug 2018 Masao Kobayakawa Shin Ohnishi Hidekazu Suzuki Gastric varices (GVs) are a major complication of portal hypertension in patients with liver cirrhosis. The mortality rate associated with the bleeding from GVs is not low. Balloon‐occluded retrograde transvenous obliteration (BRTO) was first introduced by Kanagawa et al. as a treatment for isolated GVs in 1994. It has been performed most frequently in Asia, especially in Japan. Ethanolamine oleate was the original sclerosant used in the therapy. Since the late 2000s, BRTO using sodium tetradecyl sulfate foam or polidocanol foam as a sclerosant has been performed in many countries other than Japan. Then, early in the 2010s, modified BRTO techniques including vascular plug–assisted retrograde transvenous obliteration (PARTO) and coil‐assisted retrograde transvenous obliteration (CARTO) was developed as an alternative treatment for GVs. This article provides a historical overview of BRTO using various sclerosants and modified BRTO techniques such as PARTO and CARTO. ... Itou et al. 15 demonstrated that the technical success in achieving obliteration of varices using polidocanol foam is comparable to that of EO with a lower complication rate. In addition, polidocanol was initially used as a local anesthetic, and is therefore associated with significant reduction in pain compared with EO. 15 In fact, EO was the initial sclerosant of choice, particularly in Asia; however, this agent is associated with hemolysis and release of free hemoglobin, causing renal tubular injury and renal failure, while it is also associated with the following reported side effects: pulmonary edema, cardiogenic shock, and disseminated intravascular coagulation. 16,[19][20][21] Haptoglobin (4.000 U), which binds free hemoglobin, has been used to reduce the renal toxicity of the EO, but this is not approved by the Food and Drug Administration for use in the US. ... A comprehensive review of transvenous obliteration techniques in the management of gastric varices Article Full-text available Jan 2023 DIAGN INTERV RADIOL Irfan Masood Amin Moshksar Brian Wong Arsalan Saleem Bleeding gastric varices (GVs) is a life-threatening complication of portal hypertension, with higher morbidity and mortality rates compared with bleeding esophageal varices (EVs). The endovascular techniques for the management of GVs are mainly transjugular intrahepatic portosystemic shunt (TIPS) and transvenous obliteration of the GVs. Transvenous obliteration techniques can be an alternative or an adjunct to TIPS for treatment of GVs, depending on the clinical scenario, and are less invasive than TIPS. However, these procedures are associated with increased portal pressure and related complications, mainly worsening of the EVs. In this article, the different techniques of transvenous obliteration of GVs, their indications, contraindications, and outcomes are discussed. View ... 12,13 Unlike ethanolamine oleate iopamidol (EOI), it does not cause severe hemolytic reactions; nor does it require overnight catheterization. 14, 15 Moreover, unlike the mixture of NBCA and lipiodol, it does not require rapid injection nor rapid removal of the catheter. 16 NLE might be a suitable embolic material for patients who need embolization under balloon-occlusion and who cannot tolerate the drastic hemolytic reactions typically induced by EOI because of poor general condition. ... Percutaneous transsplenic obliteration of ectopic varices following pancreaticoduodenectomy with portal vein resection and splenic vein ligation Article Full-text available Yozo Sato Nozomi Okuno Yoshitaka Inaba Left-sided portal hypertension following pancreaticoduodenectomy (PD) with portal vein resection and splenic vein ligation may cause ectopic variceal formation, potentially resulting in life-threatening bleeding. We report of a 79-year-old male suffering from severe anemia and melena after PD. Emergency endoscopy and contrast-enhanced computed tomography (CECT) revealed ectopic varices at the anastomosis site of pancreaticojejunostomy. An interventional radiology approach was preferred over surgical and endoscopic treatment because of the poor general condition and altered anatomy. In the first procedure, percutaneous transhepatic retrograde obliteration was performed using the coaxial double balloon-occlusion technique. Although hemostasis was obtained, re-bleeding occurred two months later. CECT revealed the development of another collateral pathway and the recurrence of varices. Insufficient embolization of the afferent vein was considered the cause of recurrence. Therefore, a percutaneous transsplenic approach was used, and complete embolization of varices was achieved. When transhepatic retrograde obliteration is not effective, transsplenic antegrade obliteration can be a useful therapeutic option. ... While TIPS can be thought of as decompressing the portal circulation, BRTO can be considered as a type of portal recompression therapy, owing to the increased flow that is now circulating through the portal system in anterograde direction. Therefore, there are also reports of complications of portal hypertension such as splenomegaly and ascites [8,20,32,43,49, 56, 57]. A further disadvantage of re-compression is the worsening of esophageal varices and portal hypertensive gastropathy after BRTO [8,37,58,59]. ... Procedure for Gastric Variceal Bleeding: From BRTO to PARTO to CARTO, Three Decades of Progress Chapter Jan 2020 Edward Wolfgang Lee Damian Hall Gastric varices are less common than esophageal varices. However, it is known to be associated with a poor prognosis and mortality due to its high propensity to bleed massively. There are several management options for gastric variceal bleeding including medical management, endoscopic treatment, transjugular intrahepatic portosystemic shunt, shunt surgery, and liver transplantation. However, clinical outcomes and recurrent bleeding rate of these current treatments are widely varying, and an alternative treatment method for gastric variceal bleeding is needed. In this chapter, we present a relatively novel treatment option for gastric varices called BRTO (Balloon-occluded retrograde transvenous obliteration) and its modified methods. In brief, BRTO and modified BRTO (PARTO = plug-assisted or CARTO = coil-assisted retrograde transvenous obliteration) appear safe and effective treatment options for gastric variceal bleeding. ... If repeated venograms show insufficient clot formation, B-RTO can be reattempted (119). Sclerosants that are used in this procedure are ethanolamine oleate, sodium tetradecyl sulfate, and polidocanol, and they can be used as a foam or in a liquid version (119, 120) . ... Gastroesophageal variceal bleeding - An overview of current treatment options Rosana Troskot Gastroesophageal variceal hemorrhage is the most important clinical event that results from portal hypertension. It is a lifethreatening condition that demands rapid and efficient treatment. The first step in bleeding control is hemodynamic stabilization and pharmacological treatment, which includes administration of vasoactive drugs and short-term antibiotic prophylaxis. After initial hemodynamic stabilization, endoscopic therapy should be performed. The first choice of endoscopic treatment for esophageal bleeding is endoscopic variceal ligation (EVL), or endoscopic injection sclerotherapy (EIS) if EVL cannot be performed. Several rescue therapies, such as application of balloon tamponade, a selfexpandable metal stent (SEMS), or a transjugular intrahepatic portosystemic shunt (TIPS), are available in cases of resistant variceal bleeding that cannot be controlled with endoscopic therapies. Gastric varices have a lower incidence than esophageal varices, but bleeding from gastric varices is associated with higher mortality and morbidity rates. The first-line treatment, as with esophageal variceal bleeding, is stabilization of the patient. After that, control of bleeding can be attempted. Optimal management of gastric variceal bleeding is not yet standardized due to diverse underlying pathologies and the lack of large, randomized controlled trials. Among endoscopic techniques, endoscopic variceal obturation (EVO) has been acknowledged as reliable. Among rescue therapies, balloon-occluded retrograde transvenous obliteration (B-RTO) of gastric varices and TIPS are the most common techniques. ... The serious complications associated with EO, including hemoglobinuria and renal failure, are avoided in this study. A vital difference between liquid and foam sclerosants is that foam sclerosant can be distributed evenly within the gastric variceal system, increasing the contact surface area and improving the obliteration outcome [10, 12, 13]. Hence, foam sclerosant significantly reduced the dose of sclerosant, and the procedure-related complications could be reduced accordingly. ... Efficacy and safety of balloon-occluded retrograde transvenous obliteration of gastric varices with lauromacrogol foam sclerotherapy: initial experience Article Full-text available Jul 2018 Xuefeng Luo Huaiyuan Ma Yu Jiaze Li Yang Purpose: Balloon-occluded retrograde transvenous obliteration (BRTO) is a widely-accepted treatment for gastric varices (GVs). The purpose of this study was to evaluate the efficacy and safety of BRTO with lauromacrogol foam sclerotherapy. Materials and methods: Between May 2014 and June 2015, 32 patients were treated with lauromacrogol foam. Lauromacrogol foam was made using a combination of agents, with a 2:1:1 ratio of room air: lauromacrogol: contrast media. Patients were followed up using contrast-enhanced computed tomography (CT) and endoscopy. Results: Technical success was achieved in 31 of 32 patients (96.9%). Portal vein thrombosis occurred in two patients and resolved spontaneously. No other major complications were observed. The overall mean dose of lauromacrogol used was 12.4 mL (range, 8-20 mL). Complete obliteration of GVs was confirmed in all 31 patients (100%) on follow-up CT. Seven of the 31 patients (22.6%) experienced worsening of esophageal varices. Rupture of esophageal varices occurred in three patients and was treated successfully with band ligation. Five patients (16.1%) experienced worsening of ascites and responded well to diuretic therapy. Conclusion: BRTO utilizing lauromacrogol foam appears to be a safe and useful treatment option in patients with GVs in the short term. Percutaneous sclerotherapy using a 4 F pigtail catheter and 40 milliliters of 5% ethanolamine oleate for symptomatic large hepatic cysts Article Apr 2022 DIAGN INTERV RADIOL Shiro Miyayama Masashi Yamashiro Rie Ikeda Kazuo Notsumata PURPOSE We retrospectively evaluated the efficacy of percutaneous sclerotherapy using a 4 F catheter and 40 mL of 5% ethanolamine oleate (EO) for symptomatic large hepatic cysts. METHODS Twenty-four patients, including 10 with polycystic liver disease (PLD), were eligible. The mean long- and short-axis diameters of the cyst on computed tomography (CT) were 145.0 ± 35.5 mm (range, 72-216 mm) and 110.5 ± 21.4 mm (range, 63-150 mm), respectively. After aspiration of the fluid contents using a 4 F pigtail catheter, 40 mL of 5% EO was injected into the cyst for 30 min. Then, the catheter was withdrawn after EO removal. Symptomatic relief and complications were evaluated. The percentage reductions at the early (1-3 months later) and late (at the final follow-up) responses were evaluated using an estimated cyst volume calculated by using the following formula: volume = π/6 × long-axis diameter × (short-axis diameter)2 on the maximum cross-section image on CT. Spearman's rank correlation coefficient (ρ) was used to evaluate the correlation between the pretreatment estimated cyst volume and percentage reduction of early and late responses and between the percentage reduction of the late response and length of the follow-up period after sclerotherapy. RESULTS The symptoms disappeared in 23 patients and improved in 1 patient with PLD. The mean aspirated fluid volume was 1337.8 ± 845.4 mL (range, 140-3200 mL). In 1 patient, EO injection was postponed until the second procedure was performed 40 days later due to intraperitoneal leakage of contrast material. In another patient, the EO volume was reduced to 20 mL because of a small cyst size. The mean early and late percentage reductions of the treated cyst were 52.3% ± 23.8% and 87.5% ± 20.4% (mean follow-up period: 48.0 ± 42.4 months), respectively. The symptom recurred in 2 patients with PLD and 1 underwent additional sclerotherapy 14 months later due to re-enlargement of the treated cyst. Another patient underwent transarterial embolization 5 years and 4 months later for other enlarged cysts, although the treated cyst markedly shrank. There were significant negative correlations between the pretreatment estimated cyst volume and percentage reduction of early (P = .027, ρ = - 0.46) and late (P= .007, ρ = - 0.52) responses. However, there were no significant correlations between the percentage reduction and length of the follow-up period (P = .19, ρ = 0.31). Transient pain developed in 1 patient and low-grade fever in 3. CONCLUSION Sclerotherapy using a 4 F catheter and 40 mL of 5% EO is safe and effective for symptomatic large hepatic cysts. Plug-Assisted Retrograde Transvenous Obliteration (PARTO): Anatomical Factors Determining Procedure Outcome Article Amar Mukund Karan Anandpara Ravindran Ramalingam Shiv Kumar Sarin PurposeTo study various anatomical factors determining procedure success versus failure for plug-assisted retrograde transvenous obliteration (PARTO) of gastro-lieno-renal shunt.Materials and MethodsA retrospective review of 50 patients (M:F-37:13; mean age 58.4yrs) who were planned for PARTO of gastro-lieno-renal shunt from July 2017 to July 2019 was done. Anatomical factors [shunt diameter, shunt angle, shunt orientation, left renal vein (LRV) size/calibre and distance of shunt from renal vein-IVC confluence] were analysed on a pre-procedure CT. Statistical analysis of the data was done to assess the relationship between these factors and technical success/failure.ResultsPARTO was successfully performed in 82% (n = 41). Analysis of anatomical factors showed that aneurysmal dilatation of the LRV, extreme acute/obtuse angulation and extreme antero-posterior orientation of the shunt in relation to the LRV was associated with higher probability of technical failure of PARTO. Minimum/maximum and entry point shunt diameter was not associated with procedure outcome. It was also noted that an increased distance of the shunt from the renal vein-IVC confluence favoured trans-jugular over trans-femoral venous access for PARTO.Conclusion Knowledge of various anatomical factors of gastro-lieno-renal shunt may help in deciding the access route for PARTO and may determine technical success/failure. Alternate methods like BRTO or coil-assisted obliteration (CARTO) or anterograde obliteration of the shunt via trans-hepatic/splenic route might be needed in such circumstances. Utility of Low-dose gelatin sponge particles and 5% Ethanolamine Oleate Iopamidol Mixture in Retrograde Transvenous Obliteration (GERTO) for Gastric Varices Article Feb 2020 Atsushi Jogo Akira Yamamoto Toshio Kaminoh Yukio Miki Objectives To determine the utility of low-dose gelatin sponge particles and 5% ethanolamine oleate iopamidol (EOI) mixture in retrograde transvenous obliteration (GERTO) for gastric varices. Methods Fifty-seven consecutive patients who underwent balloon-occluded retrograde transvenous obliteration (B-RTO) for gastric varices (GV) were divided into three groups with Hirota’s grade by balloon-occluded retrograde transvenous venography. Hirota’s Grade one patients were assigned to G1 group and underwent treatment with 5% EOI. Grade ≥ 2 patients prior to August 2015 were G ≥ 2 group treated with 5% EOI, and those treated thereafter were GERTO group. The amount of EOI used per unit GV volume (EOI/GV ratio), the times to embolization and recurrence rate of GV were evaluated. Results The EOI/GV ratio was 0.66 ± 0.19 in G1, 1.5 ± 0.8 in G ≥ 2, and 0.58 ± 0.23 in GERTO (G ≥ 2 vs GERTO, p < 0.0001). The times to embolization were 26.5 ± 10.5 min for G1, 39.2 ± 26.8 for G ≥ 2, and 21.4 ± 9.4 for GERTO (G ≥ 2 vs GERTO, p = 0.005). The recurrence rate wasn’t significantly different in any of the groups. Conclusions GERTO was performed in lower amount of sclerosants and in less time compared to conventional B-RTO in Hirota’s grade ≥2. Advances in knowledge Feasibility of low-dose gelatin sponge particles and 5% ethanolamine oleate iopamidol (EOI) mixture as sclerosants for gastric varices Response to Yang et al Article Edward Wolfgang Lee Stephen Kee Balloon-Occluded Retrograde Transvenous Obliteration of a Gastric Varix with the Use of an N-Butyl Cyanoacrylate–Lipiodol–Ethanol Mixture Sep 2018 J VASC INTERV RADIOL Motoki Nakai Akira Ikoma Nobuyuki Higashino Tetsuo Sonomura Evolution of Retrograde Transvenous Obliteration Techniques Aug 2018 Mihir Patel Christopher Molvar Gastric variceal hemorrhage is a life-threatening complication of portal hypertension with a poorer prognosis compared with esophageal variceal hemorrhage. The presence of an infradiaphragmatic portosystemic shunt, often a gastrorenal shunt, allows for treatment with retrograde transvenous obliteration (RTO). RTO is an evolving treatment strategy, which includes balloon-assisted RTO, plug-assisted RTO, and coil-assisted RTO, for both gastric variceal hemorrhage and hepatic encephalopathy. RTO techniques are less invasive than transjugular intrahepatic portosystemic shunt creation, with the benefit of improved hepatic function, but at the expense of increased portal pressure. This article discusses the techniques of RTO, including patient eligibility, as well as technical and clinical outcomes, including adverse events. Long-Term Outcomes of Balloon-Occluded Retrograde Transvenous Obliteration for the Treatment of Gastric Varices: A Comparison of Ethanolamine Oleate and Sodium Tetradecyl Sulfate Hee Ho Chu Minuk Kim Hyo-Cheol Kim Purpose: This study was performed to compare the long-term outcomes of balloon-occluded retrograde transvenous obliteration (BRTO) for gastric varices using ethanolamine oleate (EO) and sodium tetradecyl sulfate (STS). Methods: From January 2002 to June 2015, 142 patients underwent BRTO for the treatment of gastric varices using EO (n = 59) or STS (n = 83). We retrospectively reviewed the follow-up data related to the obliteration of gastric varices, rebleeding, and clinical complications. The cumulative recurrence rates of gastric varices after BRTO in each group were analyzed using the Kaplan-Meier method and compared using the log-rank test. Results: The median follow-up periods were 23.9 (range 0.2-170.7) months in the BRTO with EO group and 19.9 (range 0.2-84.7) months in the BRTO with STS group. Technical success was achieved in 53 of 59 (89.8%) cases in the BRTO with EO group and 80 of 83 (96.4%) cases in the BRTO with STS group. The clinical success rates were 94.9% (56/59) in the BRTO with EO group and 96.4% (80/83) in the BRTO with STS group. The cumulative 1-, 3-, and 5-year recurrence rates for gastric varices were 3.8, 9.4, and 9.4% in the BRTO with EO group and 1.3, 2.5, and 3.8% in the BRTO with STS group, respectively (p = 0.684). Conclusions: BRTO using STS has comparable long-term outcomes to BRTO using EO for gastric varices. EUS-guided lauromacrogol ablation of insulinomas: a novel treatment Shanyu Qin Yongru Liu Hongjian Ning Haixing Jiang Background and aims: EUS-guided ablation with ethanol has been used to treat insulinoma since 2006 as a minimally invasive alternative for those who are unwilling or unsuitable for surgeries. However, pancreatic fistula, pancreatitis and other adverse effects were found after the procedure in these patients. Herein, we aimed to find a novel feasible injection. Methods: Seven patients with different chief complaints were diagnosed with insulinoma by symptoms, lab results and pathology results from EUS fine needle aspiration. All the patients refused to have surgeries and were treated by EUS-guided ablation with lauromacrogol. The injection volume was calculated by tumor size. All the patients were followed up by at least 1 month to see if there is any adverse effect. Blood glucose (BG), insulin and C-peptide levels were monitored before and after the procedure. Results: Insulinoma size ranged from 0.76 cm ×0.84 cm to 3.39 cm ×1.84 cm. With a mean injection volume of 1.9 ml (range from 0.9 to 3.9 ml), all the patients showed relief in symptoms after the procedure. During the follow up, their BG, insulin and C-peptide levels went back to normal. None of the patients had any adverse effect. Conclusions: EUS-guided ablation with lauromacrogol showed good treatment results and received no adverse effect after the procedure. Hence, we consider it as an effective and safe method to treat insulinoma. Variceal bleeding is one of the major complications of portal hypertension. Gastric variceal bleeding is less common than esophageal variceal bleeding; however, it is associated with a high morbidity and mortality rate and its management is largely uncharted due to a relatively less-established literature. In the West (United States and Europe), the primary school of management is to decompress the portal circulation utilizing the transjugular intrahepatic portosystemic shunt (TIPS). In the East (Japan and South Korea), the primary school of management is to address the gastric varices (GVs) specifically by sclerosing them utilizing the balloon-occluded retrograde transvenous obliteration (BRTO) procedure. The concept (1970s), evolution, and development (1980s-1990s) of both procedures run parallel to one another; neither is newer than the other is. The difference is that one was adopted mostly by the East (BRTO), while the other has been adopted mostly by the West (TIPS). TIPS is effective in emergently controlling bleeding for GVs even though the commonly referenced studies about managing GVs with TIPS are studies with TIPS created by bare stents. However, the results have improved with the use of stent grafts for creating TIPS. Nevertheless, TIPS cannot be tolerated by patients with poor hepatic reserve. BRTO is equally effective in controlling bleeding GVs as well as significantly reducing the GV rebleed rate. But the resultant diversion of blood flow into the portal circulation, and in turn the liver, increases the risk of developing esophageal varices and ectopic varices with their potential to bleed. Unlike TIPS, the blood diversion that occurs after BRTO improves, if not preserves, hepatic function for 6-9 months post-BRTO. The authors discuss the detailed results and critique the literature, which has evaluated and remarked on both procedures. Future research prospects and speculation as to the ideal patients for each procedure are discussed. Sclerotherapy and foam sclerotherapy for varicose veins To review published evidence concerning treatment of varicose veins using ultrasound-guided foam sclerotherapy (UGFS) to assess the safety and efficacy of this treatment. Medical literature databases including MedLine, Embase and DH-DATA were searched for recent literature concerning UGFS. Papers describing the early results and later outcome have been assessed and their main findings were included in this summary. Few randomized studies have been published in this field and much of the available data come from clinical series reported by individual clinicians. It is clear that foam sclerotherapy is far more effective than liquid sclerotherapy and that ultrasound imaging allows the treatment to be delivered accurately to affected veins. There is evidence that 3% polidocanol foam is no more effective than 1% polidocanol foam. The optimum ratio of gas to liquid is 4:1, although a range of ratios is reported in the published work. There is a wide variation in the volume used as well as the method by which it is injected. The use of carbon dioxide foam reduces the systemic complications, particularly visual disturbance, as compared with air foams. Very few serious adverse events have been reported in the literature despite the widespread use of this method. Rates of recanalization of saphenous trunks following UGFS are similar to those observed after endovenous laser and endovenous RF ablation of veins, as well as the residual incompetence after surgical treatment. UGFS is a safe and effective method of treating varicose veins. The relative advantages or disadvantages of this treatment in the longer term have yet to be published. Immediate and Midterm Complications of Sclerotherapy: Report of a Prospective Multicenter Registry of 12,173 Sclerotherapy Sessions Growing interest in sclerotherapy has emphasized the need for complete knowledge of all aspects of this method. To precisely delineate the actual incidence of immediate and delayed untoward events of daily sclerotherapy. A multicenter prospective registry was established in 22 phlebology clinics to report their activity and complications. During the study period, 12,173 sessions of sclerotherapy were carried out, 5,434 with liquid, 6,395 with foam, and 344 using both. Four thousand eighty-eight (33.9%) sessions were carried out with ultrasound guidance. Forty-nine incidents or accidents (0.4%) occurred, of which 12 were with liquid and 37 with foam. These were reported during the time of the study and an additional 1-month follow-up. Most numerous were 20 cases of visual disturbances (in 19 cases, foam or air block was used); all resolved shortly, without any after-effects. A femoral vein thrombosis was the only severe adverse event in this study. This study demonstrates that sclerotherapy is a safe technique. Etude comparative de l'efficacité d'un produit sclérosant sous ses formes liquide ou mousse dans la sclérose é choguidée des crosses des grandes et petites veines saphènes Objective: We performed balloon-occluded retrograde transvenous obliteration in three consecutive patients with small gastric varices without indwelling balloon catheter occlusion. Foam of 3% sodium tetradecyl sulfate mixed with iodized oil and room air was injected retrograde through the gastrorenal shunt, followed by a thick absorbable gelatin sponge (Gelfoam, Pfizer) plug under only 10-minute balloon occlusion. Conclusion: Because complete obliteration of gastric varices was achieved in all patients without any complications, our technique is considered to be safe and effective for small gastric varices. Since the conception of balloon-occluded retrograde transvenous obliteration (BRTO) of gastric varices 25 years ago, the placement of an indwelling balloon for hours has been central to the BRTO procedure. Numerous variables and variations of the BRTO procedure have been described, including methods to reduce sclerosant, combining percutaneous transhepatic obliteration, varying sclerosant, and using multiple sclerosants within the same procedure. However, the consistent feature of BRTO has always remained the indwelling balloon. Placing an indwelling balloon over hours for the BRTO procedure is a logistical burden that taxes the interventional radiology team and hospital resources. Substituting the balloon with hardware (coils or Amplatzer vascular plugs [AVPs] or both) is technically feasible and its risks most likely correlate with gastrorenal shunt (GRS) size. The current authors use packed 0.018- or 0.035-in coils or both for small gastric variceal systems (GRS size A and B) and AVPs for GRS sizes up to size E (from size A-E). The current authors recommend an indwelling balloon (no hardware substitute) for very large gastric variceal system (GRS size F). Substituting the indwelling balloon for hardware in size F and potentially size E GRS can also be risky. The current article describes the techniques of placing up to 16-mm AVPs through balloon occlusion guide catheters and then deflating the balloon once it has been substituted with the AVPs. In addition, 22-mm AVPs can be placed through sheaths once the balloon occlusion catheters are removed to further augment the 16-mm Amplatzer occlusion. To date, there are no studies describing, let alone evaluating, the clinical feasibility of performing BRTO without indwelling balloons. The described techniques have been successfully performed by the current authors. However, the long-term safety and effectiveness of these techniques is yet to be determined. Article Aug 2011 EUR RADIOL Sun Young Choi Jong Yun Won Kyung Ah Kim Kwang-Hun Lee To evaluate the clinical safety and effectiveness of foam sclerotherapy using polidocanol for the treatment of gastric fundal varices by balloon-occluded retrograde transvenous obliteration (BRTO). From November 2005 to June 2009, foam sclerotherapy using polidocanol for the treatment of gastric fundal varices by BRTO was performed in 16 patients (male/female 11:5; age range 46-84 years, median 67 years). Foam was made of 3% polidocanol (Aethoxysklerol; Kreussler Pharma, Wiesbaden, Germany), room air, and contrast media, with a ratio of 1:2:1, respectively. The amount of polidocanol (2-24 mL; median 7 mL) depended on the volume of varices. Technical success was achieved in 15 of 16 patients (93.8%). Technical failure occurred in one patient. All patients were without pain during sclerotherapy. One patient experienced pulmonary edema after the procedure but completely recovered with medical treatment. There was no procedure-related mortality. Patients were followed by endoscopy, computed tomography, or both. Four patients were lost to follow-up. Clinical success was achieved in 10 of 11 patients (91%). Rebleeding occurred in one case during follow-up. Foam sclerotherapy using polidocanol is clinically safe and effective for the treatment of gastric fundal varices during BRTO. Long-term results of balloon-occluded retrograde transvenous obliteration for gastric variceal bleeding and risky gastric varices: A 10-year experience Aug 2008 J GASTROEN HEPATOL Tomohiko Akahoshi Makoto Hashizume Morimasa Tomikawa Yoshihiko Maehara Balloon-occluded retrograde transvenous obliteration (B-RTO) is a new alternative treatment for gastric varices (GVx), but the long-term efficacy is not known. We investigated the long-term effects of B-RTO on rebleeding, prevention of first bleeding, mortality and occurrence of risky esophageal varices (EVx). B-RTO was performed in 68 cirrhotic patients with GVx. Twenty patients had recent bleeding, transiently treated by endoscopic Histoacryl injection or balloon tamponade. Forty-eight patients had varices likely to bleed, but no bleeding. After B-RTO, the recurrent bleeding, occurrence of EVx and mortality over the long-term were evaluated. B-RTO was successfully performed in 63 of 68 patients (92.6%). Varices eradication was confirmed by endoscopy in 61 of 63 patients (96.6%). During follow up, GVx bleeding occurred in two patients (3.2%). The 8-year cumulative rebleeding rates of patients with bleeding and risky GVx were 14% and 0%, respectively. Risky EVx occurred in 10 patients (17%) and the cumulative occurrence rate was 22% in 8 years. The cumulative occurrence rate of risky EVx was higher in GVx with EVx (GOV2-GVx) compared to GVx without EVx (IGV1, P < 0.05). No ectopic variceal bleeding occurred. No patients died from variceal bleeding. Hepatocellular carcinoma was the only significant prognostic factor (P < 0.05). B-RTO is beneficial over the long-term, despite worsening EVx in some patients, because of excellent treatment efficacy and improved mortality. We believe that B-RTO can become a first-choice radical treatment following hemostasis for gastric variceal bleeding and prophylactic treatment for risky GVx. Dec 1992 CRIT CARE MED Huiling Lee Felicity H. Hawker Warwick Selby Robert Herkes To determine the factors predicting mortality from bleeding esophageal varices and to examine the possibility of an association between the development of adult respiratory distress syndrome (ARDS) and the use of ethanolamine oleate as an esophageal variceal sclerosant. Retrospective review. ICU in a teaching hospital. A total of 101 patients with endoscopically confirmed bleeding esophageal varices were admitted on 124 occasions from 1985 to 1990. Mean age was 50 +/- 13.5 (SD) yrs. There were 62 males and 39 females. Using the Child-Pugh classification, 21.8% patients were class A, 38.6% class B, and 39.6% class C. Mean ICU and hospital lengths of stay were 5.4 +/- 5.1 and 19.6 +/- 16.1 days, respectively. Mean Acute Physiology and Chronic Health Evaluation (APACHE II) score on admission was 16.5 +/- 7.6. Endoscopic variceal sclerotherapy was performed in 99 (79.8%) of 124 ICU admissions in the 101 patients. Esophageal balloon tamponade was performed in 64 (51.6%) and a vasopressin infusion was administered in 47 (37.9%) of the 124 ICU admissions. A variety of factors was studied to find predictors of mortality and the development of ARDS. Forty-eight (48.5%) of the 101 patients died during the hospital stay. Independent predictors of mortality (by stepdown logistic regression) were total volume of ethanolamine oleate injected during sclerotherapy, multiple blood transfusions, Glasgow Coma Scale score, International normalized ratio for prothrombin test, and the presence of circulatory shock on ICU admission. Age, sex, Child-Pugh score, APACHE II score, serum bilirubin, albumin, and creatinine concentrations, use of esophageal balloon tamponade or vasopressin infusion, sepsis, pneumonia, congestive cardiac failure, aspiration, and ARDS were not statistically independent predictors of outcome. There was no difference in the mortality rates for the various causes of liver disease. Pulmonary complications occurred in 44 (43.6%) patients; sepsis occurred in 31 (25%) patients. ARDS developed in 14 patients (11.3% admissions, 13.9% patients). Statistically independent predictors of ARDS were sepsis, low plasma albumin concentration, use of esophageal balloon tamponade, and more than one sclerotherapy session. The volume and type of sclerosant used were not statistically independent predictors. Outcome is poor for patients with bleeding esophageal varices requiring ICU admission and is related to the severity of liver failure, the degree of blood loss, and failure of therapy to stop the bleeding. The findings do not support an association between the use of the sclerosant ethanolamine and the development of ARDS. Polidocanol (Aethoxyskerol) for Sclerotherapy of Superficial Venules and Telangiectasias Article Mar 1989 J Dermatol Surg Oncol Peter M. Goldman The history and possible adverse sequelae of polidocanol (Aethoxysklerol) and the sclerotherapy technique used on 1500 patients in 5000 procedures is reviewed. Treatment of gastric varices by balloon occluded retrograde transvenous obliteration Article Feb 1996 Hiroshi Kanagawa Satoaki Mima Kunio Okuda Although less common than oesophageal varices in portal hypertension, gastric fundal varices carry a higher mortality rate when they rupture. They are less amenable to sclerotherapy. We have developed a minimally invasive balloon-occluded retrograde transverse obliteration (B-RTO) procedure to treat gastric fundal varices. B-RTO involves inserting a balloon catheter into an outflow shunt (gastric-renal or gastric-vena caval inferior) via the femoral or internal jugular vein. Blood flow is then blocked by inflating the balloon, and 5% ethanolamine oleate iopamidol is injected in a retrograde manner. The embolized gastric varix subsequently disappears. B-RTO was performed in 32 patients with gastric varices. Follow-up endoscopies were performed at intervals of 2-4 months for an average observation period of 14 months. Eradication of the varices has been confirmed in 31 of 32 patients. No recurrence occurred in any patients in the follow-up period. There were no significant changes in liver function after the procedure. We conclude that B-RTO is a safe and effective procedure for the treatment of gastric fundal varices. Balloon-occluded retrograde transvenous obliteration for gastric varices with gastrorenal or gastrocaval collaterals Dec 1996 Kazumitsu Koito Tsutomu Namieno T Nagakawa K. Morita The purpose of this study was to evaluate the efficacy of balloon-occluded retrograde transvenous obliteration for gastric varices with gastrorenal or gastrocaval collaterals. Thirty patients who had gastric varices with gastrorenal or gastrocaval collaterals underwent balloon-occluded retrograde transvenous obliteration. A 5-French balloon catheter was inserted in the gastrorenal collateral, gastrocaval collateral, or both, and a 5% solution of ethanolamine oleate iopamidole that contained equal amounts of ethanolamine oleate and iopamidole 300 was injected into the gastric varices. One day, 1 week, and 1 month after balloon-occluded retrograde transvenous obliteration, hepatic and renal function tests (total bilirubin, transaminase, blood ammonia, serum creatinine, and blood urea nitrogen) were done. To evaluate therapeutic efficacy, we observed the site with endoscopy every 2 weeks and obtained enhanced CT scans every month. The observation time ranged from 10 to 30 months. After balloon-occluded retrograde transvenous obliteration, gastric varices disappeared completely in all 30 cases in 4-16 weeks (mean, 10 weeks). Recurrence of gastric varices was observed in three cases (10%), which were treated with repeated balloon-occluded retrograde transvenous obliteration. Esophageal varices were aggravated in three patients (10%), who underwent successful endoscopic injection sclerotherapy. Complications of balloon-occluded retrograde transvenous obliteration were fever and hemoglobinuria, which disappeared in about 5 days. We observed no significant hepatic and renal functional damage. Balloon-occluded retrograde transvenous obliteration offers good control of gastric varices with gastrorenal or gastrocaval collaterals, even if hepatic function is poor. Retrograde Transvenous Obliteration of Gastric Varices1 Jun 1999 Shuichiro Matsumoto Masaru Tomita Michio Kono Shozo Hirota Long-term Results of Balloon-Occluded Retrograde Transvenous Obliteration for the Treatment of Gastric Varices and Hepatic Encephalopathy J VASC INTERV RADIOL Tetsuya Fukuda Shozo Hirota Kazuro Sugimura To evaluate the long-term results of balloon-occluded retrograde transvenous obliteration (B-RTO) for the treatment of gastric varices (GV) and hepatic encephalopathy. A total of 43 patients who had undergone B-RTO were evaluated, 32 with GV, two with hepatic encephalopathy, and nine with both. All but one had been consecutively followed up with gastrointestinal endoscopy for more than 1 year (3-60 months; mean, 30.44 months). Collateral veins of gastric varices were graded using balloon-occluded retrograde left adrenal venography. The relation of both worsening of esophageal varices (EV) and improved Child-Pugh score after B-RTO to the grades of collateral vein development was analyzed. The relapse-free survival and the prognostic factors for survival after B-RTO were also assessed. GV disappeared or decreased markedly in size, and hepatic encephalopathy was completely cured in all patients. Improvement in Child-Pugh score was observed in 21 patient (50.0%) 6 months after B-RTO, but in only 11 patients (25.6%) 1 year after B-RTO. Worsening of EV was seen in eight patients and was related to a worsened grade of collateral veins. Cumulative relapse-free survival rate was 90.8% at 1 year and 87.4% at 3 years after B-RTO. The most significant prognostic factor was Child-Pugh classification (relative risk: 4.16) B-RTO is a safe and effective treatment for patients with GV and hepatic encephalopathy. The most important prognostic factors are the extent of Child-Pugh classification. Balloon-Occluded Retrograde Transvenous Obliteration of Gastric Fundal Varices with Hemorrhage Article Mikiya Kitamoto Michio Imamura Koji Kamada Kazuaki Chayama This study was performed to evaluate the clinical efficacy, feasibility, and complications of balloon-occluded retrograde transvenous obliteration for patients with hemorrhage from gastric fundal varices. Between December 1994 and February 2001, 24 consecutive patients with hemorrhage from gastric fundal varices were enrolled in this study. Balloon-occluded retrograde transvenous obliteration consisted of injecting 5% ethanolamine oleate iopamidol through the outflow vessels during balloon occlusion. The treatment was performed during acute bleeding in 11 patients and electively in 13 patients. Among those patients with acute bleeding, six were treated for temporary hemostasis with balloon tamponade, and five were treated endoscopically. Cannulation into the outflow vessels was performed in 23 patients, but the balloon catheter could not be inserted in one patient who had inferior phrenic vein outflow. Complete success was obtained in 88% (21/24) of patients, and partial success was obtained in two patients. In nine of 11 patients with acute bleeding, complete success was achieved. Rebleeding from gastric varices was not observed in patients treated with complete success, whereas two patients treated partially rebled within 1 week of the treatment (rate of rebleeding, 9%). Eradication of gastric varices was obtained in all patients (n = 19) who were examined by endoscopy 3 months after the treatment. Eight patients experienced worsening of esophageal varices. These patients were treated endoscopically because of findings that suggested a risk of hemorrhage. The overall mortality rate was 4% (1/24). No damage to the kidney was observed, although 11 patients had macrohematuria. Balloon-occluded retrograde transvenous obliteration followed by any hemostatic procedure might be effective for both prophylaxis of rebleeding and eradication of gastric fundal varices, even in urgent cases.	https://www.researchgate.net/publication/281167206_Balloon-Occluded_Retrograde_Transvenous_Obliteration_for_the_Treatment_of_Gastric_Varices_Polidocanol_Foam_Versus_Liquid_Ethanolamine_Oleate
81 FR 8398 - PATH Act Changes to Section 1445 81 FR 8398 - PATH Act Changes to Section 1445 : This document contains final and temporary regulations relating to the taxation of, and withholding on, foreign persons upon certain dispositions of, and distributions with respect to, United States real property interests (USRPIs). The regulations reflect changes made by the Protecting Americans from Tax Hikes Act of 2015 (the PATH Act). In addition, the regulations update certain mailing addresses listed in regulations under sections 897 and 1445. These regulations affect certain holders of USRPIs and withholding agents that are required to withhold tax on certain dispositions of, and distributions with respect to, USRPIs. This document also requests comments on certain other aspects of the PATH Act that apply to dispositions of, and distributions with respect to, USRPIs. 81 FR 8398 - PATH Act Changes to Section 1445 DEPARTMENT OF THE TREASURY Internal Revenue Service Federal Register Volume 81, Issue 33 (February 19, 2016) Page Range 8398-8402 FR Document 2016-03421 This document contains final and temporary regulations relating to the taxation of, and withholding on, foreign persons upon certain dispositions of, and distributions with respect to, United States real property interests (USRPIs). The regulations reflect changes made by the Protecting Americans from Tax Hikes Act of 2015 (the PATH Act). In addition, the regulations update certain mailing addresses listed in regulations under sections 897 and 1445. These regulations affect certain holders of USRPIs and withholding agents that are required to withhold tax on certain dispositions of, and distributions with respect to, USRPIs. This document also requests comments on certain other aspects of the PATH Act that apply to dispositions of, and distributions with respect to, USRPIs. [Federal Register Volume 81, Number 33 (Friday, February 19, 2016)] [Rules and Regulations] [Pages 8398-8402] From the Federal Register Online [ www.thefederalregister.org ] [FR Doc No: 2016-03421] ======================================================================= ----------------------------------------------------------------------- DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Part 1 [TD 9751] RIN 1545-BN22 PATH Act Changes to Section 1445 AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Final and temporary regulations. ----------------------------------------------------------------------- SUMMARY: This document contains final and temporary regulations relating to the taxation of, and withholding on, foreign persons upon certain dispositions of, and distributions with respect to, United States real property interests (USRPIs). The regulations reflect changes made by the Protecting Americans from Tax Hikes Act of 2015 (the PATH Act). In addition, the regulations update certain mailing addresses listed in regulations under sections 897 and 1445. These regulations affect certain holders of USRPIs and withholding agents that are required to withhold tax on certain dispositions of, and distributions with respect to, USRPIs. This document also requests comments on certain other aspects of the PATH Act that apply to dispositions of, and distributions with respect to, USRPIs. DATES: Effective date: These regulations are effective on February 19, 2016. Applicability date: For dates of applicability, see Sec. Sec. 1.1445-1(h), 1.1445-2(e), and 1.1445-5(h). ADDRESSES: Send submissions to: CC:PA:LPD:PR (REG-101329-16), Room 5203, Internal Revenue Service, P.O. Box 7604, Ben Franklin Station, Washington, DC 20044. Submissions may be hand-delivered Monday through Friday between the hours of 8 a.m. and 4 p.m. to CC:PA:LPD:PR (REG- 101329-16), Courier's Desk, Internal Revenue Service, 1111 Constitution Avenue NW., Washington, DC 20224, or sent electronically via the Federal eRulemaking Portal at (IRS REG- 101329-16). FOR FURTHER INFORMATION CONTACT: Milton M. Cahn or David A. Levine, (202) 317-6937; concerning submissions of comments, Regina Johnson, (202) 317-6901 (not toll-free numbers). SUPPLEMENTARY INFORMATION: Paperwork Reduction Act The collections of information contained in these final regulations were previously reviewed and approved by the Office of Management and Budget in accordance with the Paperwork Reduction Act of 1995 (44 U.S.C. 3507) under control numbers 1545-0123, 1545-0902, and 1545-1797 in conjunction with Treasury decisions 7999 (49 FR 50689, Dec. 31, 1984), 8113 (51 FR 46620, Dec. 24, 1986), and 9082 (68 FR 46081, Aug. 5, 2003), respectively. There are no proposals for substantive changes to these collections of information. An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a valid control number assigned by the Office of Management and Budget. Books or records relating to a collection of information must be retained as long as their contents might become material in the administration of any internal revenue law. Generally, tax returns and tax return information are confidential, as required by 26 U.S.C. 6103. Background Section 897(a)(1) provides, in general, that gain or loss of a nonresident alien individual or foreign corporation from the disposition of a United States real property interest (USRPI) shall be taken into account under section 871(b)(1) or 882(a)(1), as applicable, as if the nonresident alien individual or foreign corporation were engaged in a trade or business within the United States during the taxable year and such gain or loss were effectively connected with that trade or business. Section 897(c)(1)(A) defines a USRPI to include any interest (other than solely as a creditor) in any domestic corporation unless the taxpayer establishes that such corporation was at no time a United States real property holding corporation (USRPHC) during the applicable testing period (generally, the five-year period ending on the date of the disposition of the USRPHC). Under section 897(c)(2), a USRPHC means any corporation if the fair market value of its USRPIs equals or exceeds 50 percent of the total fair market value of its USRPIs, its interests in real property located outside the United States, and any other assets that are used or held for use in a trade or business. However, section 897(c)(1)(B) generally provides [[Page 8399]] that an interest in a corporation is not a USRPI if the corporation does not hold USRPIs as of the date its stock is sold and the corporation disposed of all of the USRPIs that it held during the applicable testing period in transactions in which the full amount of gain, if any, was recognized (the cleansing exception). Section 1445(a) generally imposes a withholding tax obligation on the transferee when a foreign person disposes of a USRPI. Section 1445(f)(3) provides that a foreign person is any person other than a United States person. Section 1445(e)(3) generally imposes a withholding obligation on a domestic corporation that is a USRPHC on distributions to foreign persons to which section 302 or sections 331 through 346 apply. Section 1445(e)(3) also provides that similar rules are applicable to distributions to foreign persons under section 301 that are not made out of the earnings and profits of the domestic corporation. Section 1445(e)(4) generally requires a domestic or foreign partnership, the trustee of a domestic or foreign trust, or the executor of a domestic or foreign estate to withhold on the distribution of any USRPI to a partner or beneficiary who is a foreign person. Under section 1445(e)(5), the transferee of a partnership interest or of a beneficial interest in a trust or estate is required to deduct and withhold tax to the extent provided in regulations. Any amounts withheld under section 1445 are credited against the foreign transferor's U.S. tax liability. Sec. 1.1445-1(f)(1). Before the enactment of the Protecting Americans from Tax Hikes Act of 2015, Public Law 114-113 (the PATH Act), the withholding rate under sections 1445(a), 1445(e)(3), 1445(e)(4), and 1445(e)(5) was 10 percent of either the amount realized or the fair market value of the interest, as applicable. Section 324(a) of the PATH Act increased the withholding rate under these sections from 10 percent to 15 percent. This new rate applies to dispositions after February 16, 2016. Section 324(b) of the PATH Act, however, retained the 10-percent withholding rate in the case of a disposition of property that is acquired by the transferee for his or her use as a residence with respect to which the amount realized is greater than $300,000 but does not exceed $1 million. Section 325 of the PATH Act provides that the cleansing exception will not apply to dispositions on or after December 18, 2015, if the corporation or its predecessor was a real estate investment trust or a regulated investment company at any time during the shorter of the period that the shareholder held the interest or the five-year period ending on the date of the disposition of the shareholder's interest in the corporation. Section 323(a) of the PATH Act added section 897(l), which provides that section 897 does not apply (i) to USRPIs held directly (or indirectly through one or more partnerships) by, or (ii) to distributions received from a real estate investment trust by, a qualified foreign pension fund or an entity wholly owned by a qualified foreign pension fund. Section 897(l)(2) defines a qualified foreign pension fund for purposes of section 897(l), and section 897(l)(3) provides that the Secretary shall prescribe such regulations as may be necessary or appropriate to carry out the purposes of section 897(l). In addition, section 323(b) of the PATH Act amended the definition of foreign person in section 1445(f)(3) to provide that entities described in section 897(l) are not treated as foreign persons for purposes of section 1445, except as otherwise provided by the Secretary. The amendments in section 323 of the PATH Act are applicable to dispositions and distributions after December 18, 2015. Explanation of Provisions These regulations update Sec. 1.897-2 and Sec. Sec. 1.1445-1 through 1.1445-5, and append an informational footnote to Sec. 1.1445- 11T(d)(2)(iii), to reflect changes made by the PATH Act. Additionally, for certain filings that are described in regulations under sections 897 and 1445, these regulations provide that the mailing address is the address specified in the Instructions for Form 8288 under the heading ``Where To File.'' Applicability Dates Consistent with the PATH Act, the revisions to Sec. 1.1445-2 to incorporate the exemption under section 1445(f)(3) for entities described in section 897(l) apply to dispositions and distributions after December 18, 2015, and the revisions to Sec. 1.897-2 regarding the cleansing exception apply to dispositions on or after December 18, 2015. The new withholding rates described in these regulations apply to dispositions of, and distributions with respect to, USRPIs that occur after February 16, 2016. Beginning after February 19, 2016, taxpayers are required to use the revised mailing address provided in these regulations. However, the IRS will not assert penalties against taxpayers that use the mailing address previously specified in the regulations on or before June 20, 2016. Any prior timely filings made pursuant to the regulations under sections 897 and 1445 that were mailed to the address specified in the Instructions for Form 8288 under the heading ``Where To File,'' instead of the address previously specified in the regulations, have been accepted by the IRS. Request for Comments The Treasury Department and the IRS request comments regarding what regulations, if any, should be issued pursuant to section 897(l)(3). All comments that are submitted as prescribed in this preamble under the ADDRESSES heading will be available at www.regulations.gov or upon request. Special Analyses Certain IRS regulations, including this one, are exempt from the requirements of Executive Order 12866, as supplemented and reaffirmed by Executive Order 13563. Therefore, a regulatory impact assessment is not required. Because no notice of proposed rulemaking is required, a Regulatory Flexibility Analysis under the Regulatory Flexibility Act (5 U.S.C. chapter 6) is not required. The Treasury Department and the IRS have determined that section 553(b) of the Administrative Procedure Act (APA) (5 U.S.C. chapter 5) does not apply to these regulations, including because good cause exists under section 553(b)(B) of the APA. Section 553(b)(B) of the APA provides that an agency is not required to publish a notice of proposed rulemaking in the Federal Register when the agency, for good cause, finds that notice and public comment thereon are impracticable, unnecessary, or contrary to the public interest. These regulations are necessary to ensure that existing regulations for transferees and other parties properly reflect the changes implemented by the PATH Act. Because these regulations merely conform the regulations to certain changes made by the PATH Act, and update certain mailing addresses, prior notice and public comment is unnecessary. Accordingly, good cause exists for dispensing with notice and public comment pursuant to section 553(b) of the APA. For the same reasons that section 553(b) of the APA does not apply, including because good cause exists under section 553(d)(3) of the APA, the requirements in section 553(d) of the APA for a delayed effective date are inapplicable. Drafting Information The principal authors of these regulations are Milton M. Cahn and [[Page 8400]] David A. Levine of the Office of Associate Chief Counsel (International). However, other personnel from the IRS and the Treasury Department participated in their development. List of Subjects in 26 CFR Part 1 Income taxes, Reporting and recordkeeping requirements. Adoption of Amendments to the Regulations Accordingly, 26 CFR part 1 is amended as follows: PART 1--INCOME TAXES 0 Paragraph 1. The authority citation for part 1 continues to read in part as follows: Authority: 26 U.S.C. 7805 * * * 0 Par. 2. Section 1.897-2 is amended as follows: 0 1. By removing ``, and'' at the end of paragraph (f)(2)(i) and adding a semicolon in its place. 0 2. By removing the period at the end of paragraph (f)(2)(ii) and adding ``; and'' in its place. 0 1. By adding paragraph (f)(2)(iii) before the existing undesignated paragraph. 0 2. In each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (h)(2)(v), third sentence... the Director, the address Philadelphia specified in the Service Center, Instructions for P.O. Box 21086, Form 8288 under the Drop Point 8731, heading ``Where To FIRPTA Unit, File''. Philadelphia, PA 19114-0586. (h)(4)(ii), first sentence.. the Director, the address Philadelphia specified in the Service Center, Instructions for P.O. Box 21086, Form 8288 under the Drop Point 8731, heading ``Where To FIRPTA Unit, File''. Philadelphia, PA 19114-0586. ------------------------------------------------------------------------ The revision and addition reads as follows: Sec. 1.897-2 United States real property holding corporations. * * * * * (f) * * * (2) * * * (iii) If the disposition occurs on or after December 18, 2015, neither the corporation nor any predecessor of the corporation was a regulated investment company or a real estate investment trust at any time during the shorter of the periods described in section 897(c)(1)(A)(ii). * * * * * Sec. 1.897-3 [Amended] 0 Par. 3. Section 1.897-3 is amended in each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (c) introductory text, first with the Director, at the address sentence. Philadelphia specified in the Service Center, Instructions for P.O. Box 21086, Form 8288 under Drop Point 8731, the heading FIRPTA Unit, ``Where To Philadelphia, PA File''. 19114-0586. (d)(1), fourth sentence......... the Philadelphia the address Service Center. specified in the Instructions for Form 8288 under the heading ``Where To File''. (f)(1), second sentence......... addressed to the delivered to the Director, address specified Philadelphia in the Service Center, Instructions for P.O. Box 21086, Form 8288 under Drop Point 8731, the heading FIRPTA Unit, ``Where To Philadelphia, PA File''. 19114-0586. (f)(1), fifth sentence.......... the Director, the address Philadelphia specified in the Service Center. Instructions for Form 8288 under the heading ``Where To File''. ------------------------------------------------------------------------ 0 Par. 4. Section 1.1445-1 is amended as follows: 0 1. By revising the first sentence and removing the last sentence of paragraph (b)(1). 0 2. By redesignating paragraphs (b)(2) through (4) as paragraphs (b)(3) through (5) respectively. 0 3. By adding a new paragraph (b)(2). 0 4. By revising paragraph (g)(10). 0 5. By revising the heading and adding two sentences after the first sentence in paragraph (h). 0 6. In each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (a), second sentence........ 10 percent.......... 15 percent (10 percent in the case of dispositions described in paragraph (b)(2) of this section). newly designated 10 percent.......... 15 percent. (b)(4)(iii), first sentence. newly designated Sec. 1.1445- paragraph (b)(4)(iii), second 1(b)(3)(iii). (b)(4)(iii). sentence. (c)(2)(i)(A), first sentence 10 percent.......... 15 percent (10 percent in the case of dispositions described in paragraph (b)(2) of this section). (c)(2)(i)(B), third sentence 10 percent.......... 15 percent (10 percent in the case of dispositions described in paragraph (b)(2) of this section). ------------------------------------------------------------------------ [[Page 8401]] The additions and revisions read as follows: Sec. 1.1445-1 Withholding on dispositions of U.S. real property interests by foreign persons: In general. * * * * * (b) Duty to withhold--(1) In general. Except as provided in paragraph (b)(2) and Sec. Sec. 1.1445-2 and 1.1445-3, transferees of U.S. real property interests are required to deduct and withhold a tax equal to 15 percent of the amount realized by the transferor if the transferor is a foreign person.* * * (2) Reduced rate for certain residences. Transferees of U.S. real property interests are required to deduct and withhold a tax equal to 10 percent of the amount realized by the transferor if the transferor is a foreign person and the following requirements are satisfied: (i) The property is acquired by the transferee for use by the transferee as a residence; (ii) the amount realized for the property does not exceed $1,000,000; and (iii) section 1445(b)(5) does not apply to the disposition. See Sec. 1.1445-2(d)(1). * * * * * (g) * * * (10) Address for correspondence. Any written communication to the Internal Revenue Service described in this section is to be mailed to the address specified in the Instructions for Form 8288 under the heading ``Where To File.'' (h) Applicability dates. * * * The withholding rates set forth in paragraphs (a), (b)(1), (b)(2), (b)(4)(iii), (c)(2)(i)(A), and (c)(2)(i)(B) of this section apply to dispositions after February 16, 2016. For dispositions on or before February 16, 2016, see paragraphs (a), (b)(1), (b)(3)(iii), (c)(2)(i)(A), and (c)(2)(i)(B) of this section as contained in 26 CFR part 1 revised as of April 1, 2015. 0 Par. 5. Section 1.1445-2 is amended as follows: 0 1. By revising the first sentence in the undesignated paragraph following paragraph (b)(2)(i)(C). 0 2. In paragraph (b)(4)(iv), by adding a sentence after the last sentence. 0 3. In paragraph (e), by revising the heading and adding two sentences after the first sentence. 0 4. In each of the paragraphs listed in the first colum by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (b)(4)(iv), second sentence. 10 percent.......... 15 percent. (b)(4)(iv), third sentence.. 10 percent.......... 15 percent. (b)(4)(iv), fourth sentence. 10 percent.......... 15 percent. (c)(3)(iii), second sentence 10 percent.......... 15 percent. (c)(3)(iii), third sentence. 10 percent.......... 15 percent. (c)(3)(iii), fourth sentence 10 percent.......... 15 percent. (d)(2)(i)(B), first sentence provides a copy of mails a copy of the the transferor's transferor's notice notice to the to the Internal Director, Revenue Service. Philadelphia Service Center. (d)(3)(i)(A) introductory 10 percent.......... 15 percent (10 text, first sentence. percent in the case of dispositions described in Sec. 1.1445-1(b)(2)). (d)(3)(i)(B) introductory 10 percent.......... 15 percent (10 text, first sentence. percent in the case of dispositions described in Sec. 1.1445-1(b)(2)). ------------------------------------------------------------------------ The additions and revision read as follows: Sec. 1.1445-2 Situations in which withholding is not required under section 1445(a). * * * * * (b) * * * (2) * * * (i) * * * (C) * * * In general, a foreign person is a nonresident alien individual, foreign corporation, foreign partnership, foreign trust, or foreign estate, but not a qualified foreign pension fund (as defined in section 897(l)) or an entity all of the interests of which are held by a qualified foreign pension fund. * * * (4) * * * (iv) * * * For dispositions described in Sec. 1.1445-1(b)(2), this paragraph shall be applied by replacing ``15 percent'' with ``10 percent'' each time it appears. * * * * * (e) Applicability dates. * * * The exclusion of entities described in section 897(l) from the definition of foreign person in paragraph (b)(2)(i) of this section applies to dispositions and distributions after December 18, 2015, and the withholding rates set forth in paragraphs (b)(4)(iv), (c)(3)(iii), and (d)(3)(i) of this section apply to dispositions after February 16, 2016. For dispositions on or before February 16, 2016, see paragraphs (b)(4)(iv), (c)(3)(iii), and (d)(3)(i) of this section as contained in 26 CFR part 1 revised as of April 1, 2015. Sec. 1.1445-3 [Amended] 0 Par. 6. Section 1.1445-3 is amended in each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (b)(1), first sentence...... to the Director, to. Philadelphia Service Center, at. (f)(1), first sentence...... to the Director, to. Philadelphia Service Center, at. (f)(2)(iii), heading........ by the Director, on behalf of the Philadelphia Service. Service Center,. (f)(2)(iii), first sentence. by the Director, on behalf of the Philadelphia Service. Service Center or his delegate. (g) introductory text, third addressed to the delivered to. sentence. Director, Philadelphia Service Center, at. ------------------------------------------------------------------------ [[Page 8402]] Sec. 1.1445-4 [Amended] 0 Par. 7. Section 1.1445-4 is amended in each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (c)(1), tenth sentence...... from a foreign from a foreign person must person after withhold a tax February 16, 2016, equal to 10 percent. must withhold a tax equal to 15 percent (10 percent in the case of dispositions described in Sec. 1.1445-1(b)(2)). (c)(1), thirteenth sentence. 10 percent tax...... 15 percent tax (10 percent tax in the case of dispositions described in Sec. 1.1445-1(b)(2)). (c)(2), second sentence..... to the Director, to. Philadelphia Service Center, at. ------------------------------------------------------------------------ 0 Par. 8. Section 1.1445-5 is amended as follows: 0 1. In each of the paragraphs listed in the first column, by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (b)(2)(ii) introductory to the Director, to. text, first sentence. Philadelphia Service Center, at. (c)(3)(iv) introductory 10 percent.......... 15 percent. text, second sentence. (c)(3)(v), first sentence... with the Director, at. Philadelphia Service Center, at. (c)(3)(v), fifth sentence... with the Director, at. Philadelphia Service Center, at. (e)(1) introductory text, 10 percent.......... 15 percent. first sentence. ------------------------------------------------------------------------ 0 2. In paragraph (h), by revising the heading and adding two sentences after the first sentence. The revision and additions read as follows: Sec. 1.1445-5 Special rules concerning distributions and other transactions by corporations, partnerships, trusts, and estates. * * * * * (h) Applicability dates. * * * The withholding rates set forth in paragraphs (c)(3)(iv) and (e)(1) of this section apply to distributions after February 16, 2016. For distributions on or before February 16, 2016, see paragraphs (c)(3)(iv) and (e)(1) of this section as contained in 26 CFR part 1 revised as of April 1, 2015. Sec. 1.1445-6 [Amended] 0 Par. 9. Section 1.1445-6 is amended in each of the paragraphs listed in the first column by removing the language in the ``Remove'' column and adding in its place the language in the ``Add'' column. ------------------------------------------------------------------------ Paragraph Remove Add ------------------------------------------------------------------------ (f)(1), first sentence...... to the Director, to. Philadelphia Service Center, at. (f)(2)(iii), heading........ by the Director, on behalf of the Philadelphia Service. Service Center. (f)(2)(iii), first sentence. by the Director, on behalf of the Philadelphia Service. Service Center, or his delegate. (g) introductory text, addressed to the delivered to. second sentence. Director, Philadelphia Service Center, at. ------------------------------------------------------------------------ Sec. 1.1445-11T [Amended] 0 Par. 10. Section 1.1445-11T is amended in paragraph (d)(2)(iii) by adding footnote ``1'' after the last sentence to read as follows: Sec. 1.1445-11T Special rules requiring withholding under Sec. 1.1445-5 (temporary). * * * * * (d) * * * (2) * * * (iii) * * * \1\ Section 324(a) of the Protecting Americans from Tax Hikes Act of 2015 (Pub. L. 114-113) increased the withholding rate under section 1445(e)(5) to 15 percent, applicable to dispositions after February 16, 2016. John Dalrymple, Deputy Commissioner for Services and Enforcement. Approved: February 12, 2016. Mark J. Mazur, Assistant Secretary of the Treasury (Tax Policy). [FR Doc. 2016-03421 Filed 2-17-16; 4:15 pm] BILLING CODE 4830-01-P Category Regulatory Information Collection Federal Register sudoc Class AE 2.7: GS 4.107: AE 2.106: Publisher Office of the Federal Register, National Archives and Records Administration Section Rules and Regulations Action Final and temporary regulations. Dates Effective date: These regulations are effective on February 19, 2016. Contact Milton M. Cahn or David A. Levine, (202) 317-6937; concerning submissions of comments, Regina Johnson, (202) 317-6901 (not toll-free numbers). FR Citation 81 FR 8398 RIN Number 1545-BN22 CFR Associated Income Taxes and Reporting and Recordkeeping Requirements	https://thefederalregister.org/81-FR/8398
Subpart 33.2—Disputes and Appeals Source: 48 FR 42349, Sept. 19, 1983, unless otherwise noted. Redesignated at50 FR 2270, Jan. 15, 1985. 33.201 Definitions. As used in this subpart— Accrual of a claimmeans the date when all events, that fix the alleged liability of either the Government or the contractor and permit assertion of the claim, were known or should have been known. For liability to be fixed, some injury must have occurred. However, monetary damages need not have been incurred. Alternative dispute resolution (ADR)means any type of procedure or combination of procedures voluntarily used to resolve issues in controversy. These procedures may include, but are not limited to, conciliation, facilitation, mediation, fact-finding, minitrials, arbitration, and use of ombudsmen. Defective certificationmeans a certificate which alters or otherwise deviates from the language in 33.207(c) or which is not executed by a person authorized to bind the contractor with respect to the claim. Failure to certify shall not be deemed to be a defective certification. Issue in controversymeans a material disagreement between the Government and the contractor that ( 1 )may result in a claim or ( 2 )is all or part of an existing claim. Misrepresentation of factmeans a false statement of substantive fact, or any conduct which leads to the belief of a substantive fact material to proper understanding of the matter in hand, made with intent to deceive or mislead. [ 48 FR 42349 , Sept. 19, 1983. Redesignated and amended at 50 FR 2270 , Jan. 15, 1985; 56 FR 67417 , Dec. 30, 1991; 59 FR 11381 , Mar. 10, 1994; 60 FR 48230 , Sept. 18, 1995; 63 FR 58594 , Oct. 30, 1998; 66 FR 2132 , Jan. 10, 2001; 67 FR 43514 , June 27, 2002; 79 FR 24212 , Apr. 29, 2014] 33.202 Disputes. 41 U.S.C. chapter 71, Disputes, establishes procedures and requirements for asserting and resolving claims subject to the Disputes statute. In addition, the Disputes statute provides for— (a) the payment of interest on contractor claims; (b) certification of contractor claims; and (c) a civil penalty for contractor claims that are fraudulent or based on a misrepresentation of fact. [56 FR 67417, Dec. 30, 1991, as amended at59 FR 11381, Mar. 10, 1994;79 FR 24212, Apr. 29, 2014] 33.203 Applicability. ( a )Except as specified in paragraph (b) below, this part applies to any express or implied contract covered by the Federal Acquisition Regulation. ( b ) This subpart does not apply to any contract with ( 1 )A foreign government or agency of that government; or ( 2 )an international organization or a subsidiary body of that organization, if the agency head determines that the application of the Disputes statute to the contract would not be in the public interest. ( c ) This part applies to all disputes with respect to contracting officer decisions on matters “arising under” or “relating to” a contract. Agency Boards of Contract Appeals (BCAs) authorized under the Disputes statute continue to have all of the authority they possessed before the Disputes statute with respect to disputes arising under a contract, as well as authority to decide disputes relating to a contract. The clause at 52.233–1, Disputes, recognizes the “all disputes” authority established by the Disputes statute and states certain requirements and limitations of the Disputes statute for the guidance of contractors and contracting agencies. The clause is not intended to affect the rights and obligations of the parties as provided by the Disputes statute or to constrain the authority of the statutory agency BCAs in the handling and deciding of contractor appeals under the Disputes statute. [ 48 FR 42349 , Sept. 19, 1983. Redesignated and amended at 50 FR 2270 , Jan. 15, 1985; 79 FR 24212 , Apr. 29, 2014] 33.204 Policy. The Government's policy is to try to resolve all contractual issues in controversy by mutual agreement at the contracting officer's level. Reasonable efforts should be made to resolve controversies prior to the submission of a claim. Agencies are encouraged to use ADR procedures to the maximum extent practicable. Certain factors, however, may make the use of ADR inappropriate (see5 U.S.C. 572(b)). Except for arbitration conducted pursuant to the Administrative Dispute Resolution Act (ADRA), (5 U.S.C. 571, et seq.), agencies have authority which is separate from that provided by the ADRA to use ADR procedures to resolve issues in controversy. Agencies may also elect to proceed under the authority and requirements of the ADRA. [59 FR 11381, Mar. 10, 1994, as amended at63 FR 58595, Oct. 30, 1998] 33.205 Relationship of the Disputes statute to Pub. L. 85–804. ( a ) Requests for relief under Pub. L. 85–804 ( 50 U.S.C. 1431 –1435) are not claims within the Disputes statute or the Disputes clause at 52.233–1, Disputes, and shall be processed under Subpart 50.1, Extraordinary Contractual Actions. However, relief formerly available only under Pub. L. 85–804; i.e., legal entitlement to rescission or reformation for mutual mistake, is now available within the authority of the contracting officer under the Disputes statute and the Disputes clause. In case of a question whether the contracting officer has authority to settle or decide specific types of claims, the contracting officer should seek legal advice. ( b ) A contractor's allegation that it is entitled to rescission or reformation of its contract in order to correct or mitigate the effect of a mistake shall be treated as a claim under the Dispute statute. A contract may be reformed or rescinded by the contracting officer if the contractor would be entitled to such remedy or relief under the law of Federal contracts. Due to the complex legal issues likely to be associated with allegations of legal entitlement, contracting officers shall make written decisions, prepared with the advice and assistance of legal counsel, either granting or denying relief in whole or in part. ( c ) A claim that is either denied or not approved in its entirety under paragraph (b) above may be cognizable as a request for relief under Pub. L. 85–804 as implemented by subpart 50.1. However, the claim must first be submitted to the contracting officer for consideration under the Disputes statute because the claim is not cognizable under Public Law 85–804, as implemented by subpart 50.1, unless other legal authority in the agency concerned is determined to be lacking or inadequate. [ 48 FR 42349 , Sept. 19, 1983, as amended at 72 FR 63030 , Nov. 7, 2007; 79 FR 24212 , Apr. 29, 2014] 33.206 Initiation of a claim. ( a ) Contractor claims shall be submitted, in writing, to the contracting officer for a decision within 6 years after accrual of a claim, unless the contracting parties agreed to a shorter time period. This 6-year time period does not apply to contracts awarded prior to October 1, 1995. The contracting officer shall document the contract file with evidence of the date of receipt of any submission from the contractor deemed to be a claim by the contracting officer. ( b ) The contracting officer shall issue a written decision on any Government claim initiated against a contractor within 6 years after accrual of the claim, unless the contracting parties agreed to a shorter time period. The 6-year period shall not apply to contracts awarded prior to October 1, 1995, or to a Government claim based on a contractor claim involving fraud. [60 FR 48230, Sept. 18, 1995] 33.207 Contractor certification. ( a )Contractors shall provide the certification specified inparagraph (c)of this section when submitting any claim exceeding $100,000. ( b )The certification requirement does not apply to issues in controversy that have not been submitted as all or part of a claim. ( c )The certification shall state as follows: I certify that the claim is made in good faith; that the supporting data are accurate and complete to the best of my knowledge and belief; that the amount requested accurately reflects the contract adjustment for which the contractor believes the Government is liable; and that I am duly authorized to certify the claim on behalf of the contractor. ( d )The aggregate amount of both increased and decreased costs shall be used in determining when the dollar thresholds requiring certification are met (see example in 15.403–4(a)(1)(iii) regarding certified cost or pricing data). ( e )The certification may be executed by any person authorized to bind the contractor with respect to the claim. ( f )A defective certification shall not deprive a court or an agency BCA of jurisdiction over that claim. Prior to the entry of a final judgment by a court or a decision by an agency BCA, however, the court or agency BCA shall require a defective certification to be corrected. [ 59 FR 11381 , Mar. 10, 1994, as amended at 60 FR 48218 , 48230 , Sept. 18, 1995; 62 FR 51271 , Sept. 30, 1997; 63 FR 58595 , Oct. 30, 1998; 75 FR 53149 , Aug. 30, 2010; 79 FR 24212 , Apr. 29, 2014] 33.208 Interest on claims. ( a ) The Government shall pay interest on a contractor's claim on the amount found due and unpaid from the date that— ( 1 )The contracting officer receives the claim (certified if required by 33.207(a)); or ( 2 )Payment otherwise would be due, if that date is later, until the date of payment. ( b ) Simple interest on claims shall be paid at the rate, fixed by the Secretary of the Treasury as provided in the Disputes statute, which is applicable to the period during which the contracting officer receives the claim and then at the rate applicable for each 6-month period as fixed by the Treasury Secretary during the pendency of the claim. (See the clause at 52.232–17 for the right of the Government to collect interest on its claims against a contractor). ( c ) With regard to claims having defective certifications, interest shall be paid from either the date that the contracting officer initially receives the claim or October 29, 1992, whichever is later. However, if a contractor has provided a proper certificate prior to October 29, 1992, after submission of a defective certificate, interest shall be paid from the date of receipt by the Government of a proper certificate. [ 59 FR 11381 , Mar. 10, 1994, as amended at 60 FR 48230 , Sept. 18, 1995; 73 FR 54005 , Sept. 17, 2008; 79 FR 24212 , Apr. 29, 2014] 33.209 Suspected fraudulent claims. If the contractor is unable to support any part of the claim and there is evidence that the inability is attributable to misrepresentation of fact or to fraud on the part of the contractor, the contracting officer shall refer the matter to the agency official responsible for investigating fraud. 33.210 Contracting officer's authority. Except as provided in this section, contracting officers are authorized, within any specific limitations of their warrants, to decide or resolve all claims arising under or relating to a contract subject to the Disputes statute. In accordance with agency policies and 33.214, contracting officers are authorized to use ADR procedures to resolve claims. The authority to decide or resolve claims does not extend to— ( a )A claim or dispute for penalties or forfeitures prescribed by statute or regulation that another Federal agency is specifically authorized to administer, settle, or determine; or ( b )The settlement, compromise, payment or adjustment of any claim involving fraud. [ 48 FR 42349 , Sept. 19, 1983. Redesignated and amended at 50 FR 2270 , Jan. 15, 1985; 51 FR 36972 , Oct. 16, 1986; 59 FR 11381 , Mar. 10, 1994; 79 FR 24212 , Apr. 29, 2014] 33.211 Contracting officer's decision. ( a ) When a claim by or against a contractor cannot be satisfied or settled by mutual agreement and a decision on the claim is necessary, the contracting officer shall— ( 1 )Review the facts pertinent to the claim; ( 2 )Secure assistance from legal and other advisors; ( 3 )Coordinate with the contract administration officer or contracting office, as appropriate; and ( 4 ) Prepare a written decision that shall include— ( i )A description of the claim or dispute; ( ii )A reference to the pertinent contract terms; ( iii )A statement of the factual areas of agreement and disagreement; ( iv )A statement of the contracting officer's decision, with supporting rationale; ( v )Paragraphs substantially as follows: “This is the final decision of the Contracting Officer. You may appeal this decision to the agency board of contract appeals. If you decide to appeal, you must, within 90 days from the date you receive this decision, mail or otherwise furnish written notice to the agency board of contract appeals and provide a copy to the Contracting Officer from whose decision this appeal is taken. The notice shall indicate that an appeal is intended, reference this decision, and identify the contract by number. With regard to appeals to the agency board of contract appeals, you may, solely at your election, proceed under the board's— (1) Small claim procedure for claims of $50,000 or less or, in the case of a small business concern (as defined in the Small Business Act and regulations under that Act), $150,000 or less; or (2) Accelerated procedure for claims of $100,000 or less. Instead of appealing to the agency board of contract appeals, you may bring an action directly in the United States Court of Federal Claims (except as provided in41 U.S.C. 7102(d), regarding Maritime Contracts) within 12 months of the date you receive this decision” ( vi )Demand for payment prepared in accordance with 32.604 and 32.605) in all cases where the decision results in a finding that the contractor is indebted to the Government. ( b )The contracting officer shall furnish a copy of the decision to the contractor by certified mail, return receipt requested, or by any other method that provides evidence of receipt. This requirement shall apply to decisions on claims initiated by or against the contractor. ( c ) The contracting officer shall issue the decision within the following statutory time limitations: ( 1 )For claims of $100,000 or less, 60 days after receiving a written request from the contractor that a decision be rendered within that period, or within a reasonable time after receipt of the claim if the contractor does not make such a request. ( 2 )For claims over $100,000, 60 days after receiving a certified claim; provided, however,that if a decision will not be issued within 60 days, the contracting officer shall notify the contractor, within that period, of the time within which a decision will be issued. ( d )The contracting officer shall issue a decision within a reasonable time, taking into account— ( 1 )The size and complexity of the claim; ( 2 )The adequacy of the contractor's supporting data; and ( 3 )Any other relevant factors. ( e )The contracting officer shall have no obligation to render a final decision on any claim exceeding $100,000 which contains a defective certification, if within 60 days after receipt of the claim, the contracting officer notifies the contractor, in writing, of the reasons why any attempted certification was found to be defective. ( f )In the event of undue delay by the contracting officer in rendering a decision on a claim, the contractor may request the tribunal concerned to direct the contracting officer to issue a decision in a specified time period determined by the tribunal. ( g )Any failure of the contracting officer to issue a decision within the required time periods will be deemed a decision by the contracting officer denying the claim and will authorize the contractor to file an appeal or suit on the claim. ( h )The amount determined payable under the decision, less any portion already paid, should be paid, if otherwise proper, without awaiting contractor action concerning appeal. Such payment shall be without prejudice to the rights of either party. [ 48 FR 42349 , Sept. 19, 1983. Redesignated at 50 FR 2270 , Jan. 15, 1985, and amended at 54 FR 34755 , Aug. 21, 1989; 59 FR 11382 , Mar. 10, 1994; 60 FR 48230 , Sept. 18, 1995; 73 FR 21800 , Apr. 22, 2008; 73 FR 54005 , Sept. 17, 2008; 79 FR 24212 , Apr. 29, 2014] 33.212 Contracting officer's duties upon appeal. To the extent permitted by any agency procedures controlling contacts with agency BCA personnel, the contracting officer shall provide data, documentation, information, and support as may be required by the agency BCA for use on a pending appeal from the contracting officer's decision. 33.213 Obligation to continue performance. ( a ) In general, before passage of the Disputes statute, the obligation to continue performance applied only to claims arising under a contract. However, the Disputes statute, at 41 U.S.C. 7103(g) , authorizes agencies to require a contractor to continue contract performance in accordance with the contracting officer's decision pending a final resolution of any claim arising under, or relating to, the contract. (A claim arising under a contract is a claim that can be resolved under a contract clause, other than the clause at 52.233–1, Disputes, that provides for the relief sought by the claimant; however, relief for such claim can also be sought under the clause at 52.233–1. A claim relating to a contract is a claim that cannot be resolved under a contract clause other than the clause at 52.233–1.) This distinction is recognized by the clause with its Alternate I (see 33.215). ( b ) In all contracts that include the clause at 52.233–1, Disputes, with its Alternate I, in the event of a dispute not arising under, but relating to, the contract, the contracting officer shall consider providing, through appropriate agency procedures, financing of the continued performance; provided, that the Government's interest is properly secured. [ 48 FR 42349 , Sept. 19, 1983. Redesignated at 50 FR 2270 , Jan. 15, 1985, as amended at 64 FR 72451 , Dec. 27, 1999; 67 FR 43514 , June 27, 2002; 79 FR 24212 , Apr. 29, 2014] 33.214 Alternative dispute resolution (ADR). ( a ) The objective of using ADR procedures is to increase the opportunity for relatively inexpensive and expeditious resolution of issues in controversy. Essential elements of ADR include— ( 1 )Existence of an issue in controversy; ( 2 )A voluntary election by both parties to participate in the ADR process; ( 3 )An agreement on alternative procedures and terms to be used in lieu of formal litigation; and ( 4 )Participation in the process by officials of both parties who have the authority to resolve the issue in controversy. ( b ) If the contracting officer rejects a contractor's request for ADR proceedings, the contracting officer shall provide the contractor a written explanation citing one or more of the conditions in 5 U.S.C. 572(b) or such other specific reasons that ADR procedures are inappropriate for the resolution of the dispute. In any case where a contractor rejects a request of an agency for ADR proceedings, the contractor shall inform the agency in writing of the contractor's specific reasons for rejecting the request. ( c ) ADR procedures may be used at any time that the contracting officer has authority to resolve the issue in controversy. If a claim has been submitted, ADR procedures may be applied to all or a portion of the claim. When ADR procedures are used subsequent to the issuance of a contracting officer's final decision, their use does not alter any of the time limitations or procedural requirements for filing an appeal of the contracting officer's final decision and does not constitute a reconsideration of the final decision. ( d )When appropriate, a neutral person may be used to facilitate resolution of the issue in controversy using the procedures chosen by the parties. ( e )The confidentiality of ADR proceedings shall be protected consistent with5 U.S.C. 574. ( f ) ( 1 )A solicitation shall not require arbitration as a condition of award, unless arbitration is otherwise required by law. Contracting officers should have flexibility to select the appropriate ADR procedure to resolve the issues in controversy as they arise. ( 2 )An agreement to use arbitration shall be in writing and shall specify a maximum award that may be issued by the arbitrator, as well as any other conditions limiting the range of possible outcomes. ( g )Binding arbitration, as an ADR procedure, may be agreed to only as specified in agency guidelines. Such guidelines shall provide advice on the appropriate use of binding arbitration and when an agency has authority to settle an issue in controversy through binding arbitration. [ 56 FR 67417 , Dec. 30, 1991, as amended at 59 FR 11382 , Mar. 10, 1994; 60 FR 48230 , Sept. 18, 1995; 63 FR 58595 , Oct. 30, 1998] 33.215 Contract clauses. ( a )Insert the clause at 52.233–1, Disputes, in solicitations and contracts, unless the conditions in 33.203(b) apply. If it is determined under agency procedures that continued performance is necessary pending resolution of any claim arising under or relating to the contract, the contracting officer shall use the clause with its Alternate I. ( b )Insert the clause at 52.233–4 in all solicitations and contracts. [69 FR 59700, Oct. 5, 2004]	https://www.ecfr.gov/api/renderer/v1/content/enhanced/2023-05-31/title-48?chapter=1&part=33&subchapter=E&subpart=33.2
Press release: Central bank digital currencies herald a new chapter for the monetary system Press Release on the pre-release of one chapter of the Annual Economic Report of the BIS, 23 June 2021. Central bank digital currencies (CBDCs) open a new chapter for money grounded on trust in the central bank. As a technologically advanced representation of money, a CBDC can further the public interest. CBDCs function best as part of a two-tier system, with the bulk of the customer-facing activities taken on by banks and other payment service providers. The most promising design for general use is a CBDC built on a digital identity scheme, safeguarding data privacy while offering protection against illicit activity and potentially streamlining cross-border payments. Central bank digital currencies herald a new chapter for the monetary system Central bank digital currencies (CBDCs) open a new chapter for money grounded on trust in the central bank. As a technologically advanced representation of money, a CBDC can further the public interest. CBDCs function best as part of a two-tier system, with the bulk of the customer-facing activities taken on by banks and other payment service providers. The most promising design for general use is a CBDC built on a digital identity scheme, safeguarding data privacy while offering protection against illicit activity and potentially streamlining cross-border payments. Central bank digital currencies are moving from concept to practical design and renew the institution of money in a new form designed for the digital age, writesthe Bank for International Settlements in its Annual Economic Report 2021. CBDCs would build on the central bank's traditional roles in the payment system, to ensure that payments are final and certain; that there is enough liquidity for the payment system to function; and that the playing field is level, by making central bank money available on an equal basis to all parties. CBDCs are a concept whose time has come. They open a new chapter for the monetary system by providing a technologically advanced representation of central bank money. In doing so, they preserve the core features of money that only the central bank can provide, anchored in the foundation of trust in the central bank. Hyun Song Shin, Economic Adviser and Head of Research of the BIS BIS analysis finds that CBDCs would best function as part of a two-tier system where the central bank and the private sector work together to do what each does well. The central bank would operate the core of the system and ensure its safety and efficiency, while the private sector, such as banks and payment service providers, would use its innovative capacity to serve customers. From a practical perspective, the BIS says the most promising CBDC design would be one tied to a digital identity, requiring users to identify themselves to access funds. A careful design would balance protecting users against the abuse of personal data with protecting the payment system against money laundering and financial crime. CBDCs could form the backbone of a new digital payment system by enabling broad access and providing strong data governance and privacy standards. They are the best way to promote the public interest case for digital money. Benoît Cœuré, Head of the BIS Innovation Hub In addition, the BIS says international cooperation on design will be vital if central banks are to harness the full benefits of CBDCs, and to improve cross-border payments while countering foreign currency substitution. The special chapter also includes analyses on: The architecture of CBDCs and how they would fit into the financial ecosystem; The international dimension of CBDC issuance and the implications for cross-border payments; Project Helvetia, which demonstrated the feasibility of integrating tokenised assets and central bank money; Application programming interfaces and how they could act as a bridge between different providers and simplify transactions. This chapteris released on 23 June, followed by the full BIS Annual Economic Report 2021, and the BIS Annual Report 2020/21, on 29 June.	https://www.bis.org/press/p210623.htm
Online machine learning for auto-scaling in the edge computing \| Request PDF Request PDF \| Online machine learning for auto-scaling in the edge computing \| The evolution of edge computing devices has enabled machine intelligence techniques to process data close to its producers (the sensors) and... \| Find, read and cite all the research you need on ResearchGate December 2022 Pervasive and Mobile Computing 87(4):101722 DOI: 10.1016/j.pmcj.2022.101722 Authors: Thiago Pereira da Silva Universidade Federal do Rio Grande do Norte Aluizio Rocha Neto Universidade Federal do Rio Grande do Norte Thaís Vasconcelos Batista Universidade Federal do Rio Grande do Norte Flávia C. Delicato Flávia C. Delicato This person is not on ResearchGate, or hasn't claimed this research yet. Show all 6 authors Hide Request full-text Download citation Copy link Link copied Request full-text Download citation Copy link Link copied To read the full-text of this research, you can request a copy directly from the authors. Citations (2) References (35) Abstract The evolution of edge computing devices has enabled machine intelligence techniques to process data close to its producers (the sensors) and end-users. Although edge devices are usually resource-constrained, the distribution of processing services among several nodes enables a processing capacity similar to cloud environments. However, the edge computing environment is highly dynamic, impacting the availability of nodes in the distributed system. In addition, the processing workload for each node can change constantly. Thus, the scaling of processing services needs to be rapidly adjusted, avoiding bottlenecks or wasted resources while meeting the applications’ QoS requirements. This paper presents an auto-scaling subsystem for container-based processing services using online machine learning. The auto-scaling follows the MAPE-K control loop to dynamically adjust the number of containers in response to workload changes. We designed the approach for scenarios where the number of processing requests is unknown beforehand. We developed a hybrid auto-scaling mechanism that behaves reactively while a prediction online machine learning model is continuously trained. When the prediction model reaches a desirable performance, the auto-scaling acts proactively, using predictions to anticipate scaling actions. An experimental evaluation has demonstrated the feasibility of the architecture. Our solution achieved fewer service level agreement (SLA) violations and scaling operations to meet demand than purely reactive and no scaling approaches using an actual application workload. Also, our solution wasted fewer resources compared to the other techniques. Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations Join for free No full-text available To read the full-text of this research, you can request a copy directly from the authors. Request full-text PDF Citations (2) References (35) ... Therefore, the scaling of processing services must be dynamic. Recent work has studied online machine learning for autoscaling, such as the one in [150] , in which the authors present an autoscaling subsystem for container-based processing services. However, it will be interesting and promising to design dynamic autoscaling to ensure the scalability of the system with high QoS performance. ... At the Confluence of Artificial Intelligence and Edge Computing in IoT-Based Applications: A Review and New Perspectives Article Full-text available Feb 2023 SENSORS-BASEL Amira Bourechak Ouarda Zedadra Med Nadjib Kouahla Giancarlo Fortino Given its advantages in low latency, fast response, context-aware services, mobility, and privacy preservation, edge computing has emerged as the key support for intelligent applications and 5G/6G Internet of things (IoT) networks. This technology extends the cloud by providing intermediate services at the edge of the network and improving the quality of service for latency-sensitive applications. Many AI-based solutions with machine learning, deep learning, and swarm intelligence have exhibited the high potential to perform intelligent cognitive sensing, intelligent network management, big data analytics, and security enhancement for edge-based smart applications. Despite its many benefits, there are still concerns about the required capabilities of intelligent edge computing to deal with the computational complexity of machine learning techniques for big IoT data analytics. Resource constraints of edge computing, distributed computing, efficient orchestration, and synchronization of resources are all factors that require attention for quality of service improvement and cost-effective development of edge-based smart applications. In this context, this paper aims to explore the confluence of AI and edge in many application domains in order to leverage the potential of the existing research around these factors and identify new perspectives. The confluence of edge computing and AI improves the quality of user experience in emergency situations, such as in the Internet of vehicles, where critical inaccuracies or delays can lead to damage and accidents. These are the same factors that most studies have used to evaluate the success of an edge-based application. In this review, we first provide an in-depth analysis of the state of the art of AI in edge-based applications with a focus on eight application areas: smart agriculture, smart environment, smart grid, smart healthcare, smart industry, smart education, smart transportation, and security and privacy. Then, we present a qualitative comparison that emphasizes the main objective of the confluence, the roles and the use of artificial intelligence at the network edge, and the key enabling technologies for edge analytics. Then, open challenges, future research directions, and perspectives are identified and discussed. Finally, some conclusions are drawn. View Show abstract Auto-scaling techniques in container-based cloud and edge/fog computing: Taxonomy and survey Article Jun 2023 COMPUT COMMUN Javad Dogani Reza Namvar Farshad Khunjush View A cost-efficient auto-scaling mechanism for IoT applications in fog computing environment: a deep learning-based approach Article Full-text available Dec 2021 CLUSTER COMPUT Masoumeh Etemadi Mostafa Ghobaei-Arani Ali Shahidinejad The fog computing model has emerged as a viable infrastructure for serving IoT applications in recent years. In the fog ecosystem, it is essential to manage resources for different workloads due to the high volume and rapid growth of requests. Therefore, a challenge faced in this area is dynamic and efficient resource auto-scaling because fog resources must be allocated to requests efficiently. More fog resources than needed leads to “Over-Provisioning”, and fewer fog resources leads to the “Under-provisioning” issue. To this end, an effective deep learning-based resource auto-scaling mechanism has been proposed to manage the number of resources needed to handle dynamic workloads in a fog environment. The simulation results indicated that the proposed solution reduces cost, network usage, and delay violation and increases CPU utilization compared with existing resource auto-scaling mechanisms. View Show abstract Leveraging Edge Intelligence for Video Analytics in Smart City Applications Article Full-text available Dec 2020 Thiago Pereira da Silva Thaís Vasconcelos Batista Aluizio Rocha Neto Frederico Lopes In smart city scenarios, the huge proliferation of monitoring cameras scattered in public spaces has posed many challenges to network and processing infrastructure. A few dozen cameras are enough to saturate the city’s backbone. In addition, most smart city applications require a real-time response from the system in charge of processing such large-scale video streams. Finding a missing person using facial recognition technology is one of these applications that require immediate action on the place where that person is. In this paper, we tackle these challenges presenting a distributed system for video analytics designed to leverage edge computing capabilities. Our approach encompasses architecture, methods, and algorithms for: (i) dividing the burdensome processing of large-scale video streams into various machine learning tasks; and (ii) deploying these tasks as a workflow of data processing in edge devices equipped with hardware accelerators for neural networks. We also propose the reuse of nodes running tasks shared by multiple applications, e.g., facial recognition, thus improving the system’s processing throughput. Simulations showed that, with our algorithm to distribute the workload, the time to process a workflow is about 33% faster than a naive approach. View Show abstract A Systematic Review on Software Architectures for IoT Systems and Future Direction to the Adoption of Microservices Architecture Article Full-text available Nov 2020 Abdul Razzaq The Internet of Things-based systems and software allow computations anywhere at any time by interconnecting individuals, networks, services, computers and artefacts that allow autonomous systems to form digitized communities. As the blueprint for software-intensive applications, and software architecture that precise the complexity of a network's planning, development , and changing phases to effectively and efficiently build complex IoT-driven applications. In any case, there exists no comprehensive analysis in the state of the research on the adoption of MSA for IoT systems. This study effort is needed to explore architectural concepts and practices for designing and developing IoT software to excel state-of-the-art for IoTs along with suggestions and recommendations for IoT software to the adoption of MSA to fulfil the identified gaps. A systematic analysis was coordinated, covering up the literature on existing IoT solutions by studying 140 qualitatively selected articles performed between 2005 and Jan 2020. One hundred forty articles were comprised in this SLR. The findings of this study demonstrated different research topics including software architectural styles, patterns, and models to build IoT software. This research presents cloud-based computing environments, autonomous, software-defined networking, and responsive applications, and IoT-driven agent-based systems, (1) thirteen MSA architectural and design patterns for IoTs and classification of patterns, (2) classification of software architectures for IoTs into nine main categories and their sub-categories, (3) twenty-three most investigated IoT challenges, and (4) mapping of IoT challenges with software architectural solutions. The study revealed the innovative work on IoT software architecture and trends that help in the creation and dynamic adaptation of IoT software for reusability, automation and human decision-making. The outputs of this SLR are useful in revealing many recommendations to the software industry, software engineering community, and computer sciences community with over the past 15 years of research into the adoption of MSA. This study reflects a distilled awareness of architectural practices and principles to assist researchers and practitioners in promoting information sharing software architectural roles and responsibilities for the Internet of Things software. View Show abstract An Architecture for Distributed Video Stream Processing in IoMT Systems Article Full-text available Aug 2020 Aluizio Rocha Neto Thiago Pereira da Silva Thaís Vasconcelos Batista Frederico Lopes In Internet of Multimedia Things (IoMT) systems, Internet cameras installed in buildings and streets are major sources of sensing data. From these large-scale video streams, it is possible to infer various information providing the current status of the monitored environments. Some events of interest that have occurred in these observed locations produce insights that might demand near real-time responses from the system. In this context, the event processing depends on data freshness, and computation time, otherwise, the processing results and activities become less valuable or even worthless. An encouraging plan to support the computational demand for latency-sensitive applications of largely geo-distributed systems is applying Edge Computing resources to perform the video stream processing stages. However, some of these stages use deep learning methods for the detection and identification of objects of interest, which are voracious consumers of computational resources. To address these issues, this work proposes an architecture to distribute the video stream processing stages in multiple tasks running on different edge nodes, reducing network overhead and consequent delays. The Multilevel Information Fusion Edge Architecture (MELINDA) encapsulates the data analytics algorithms provided by machine learning methods in different types of processing tasks organized by multiple data-abstraction levels. This distribution strategy, combined with the new category of Edge AI hardware specifically designed to develop smart systems, is a promising approach to address the resource limitations of edge devices. View Show abstract Rule based auto-scalability of IoT services for efficient edge device resource utilization Article Full-text available Dec 2020 Ahmed Bali Mahmud Al-Osta Soufiene Ben Dahsen Abdelouahed Gherbi Conveying the workload of IoT systems from the cloud to edge nodes have been widely adopted by industrial and academic sectors. This tendency is generally promoted to meet the requirements of some time-sensitive use cases such as IoT healthcare applications. However, IoT devices at the edge network are likely to be resource-limited, as well as, they perform under an extremely heterogeneous environment in terms of the connected devices and the deployed software modules. Thus, both of the aforementioned concerns have considerably led to hindering the deployment process of services on IoT edge devices. In this paper, we propose an approach to facilitate a scalable and lightweight solution for service deployment for efficient resource utilization on IoT edge nodes. Our solution is based on the container concept, and we adopt the cluster concept to define a group of IoT edge devices. Containers are lightweight virtualization technique that enables services to be packaged and deployed with their dependencies regardless of the hosts infrastructure, as well as, they facilitate the service communication and the update process. Furthermore, containers are supported by some means of orchestration such as swarm. These orchestration tools can be configured to enable services deployment and resources sharing among IoT edge devices falling within the same cluster. However, they lack elasticity in terms of auto-scaling up/down of services instances in corresponding to the resource utilization of all cluster elements, as well as, service performance metrics. Our approach overcomes these limitations by following an auto-scaling process based on MAPE-K loop, which is based on our proposed rule model to generate a scaling plan by analyzing collected performance metrics of a cluster. Our evaluation shows the efficiency of the proposed approach in adapting the system performance to meet service performance requirements and the availability of system resources. View Show abstract Machine learning for streaming data: state of the art, challenges, and opportunities Article Full-text available Nov 2019 Heitor Murilo Gomes Jesse Read Albert Bifet Joao Gama Incremental learning, online learning, and data stream learning are terms commonly associated with learning algorithms that update their models given a continuous influx of data without performing multiple passes over data. Several works have been devoted to this area, either directly or indirectly as characteristics of big data processing, i.e., Velocity and Volume. Given the current industry needs, there are many challenges to be addressed before existing methods can be efficiently applied to real-world problems. In this work, we focus on elucidating the connections among the current stateof- the-art on related fields; and clarifying open challenges in both academia and industry. We treat with special care topics that were not thoroughly investigated in past position and survey papers. This work aims to evoke discussion and elucidate the current research opportunities, highlighting the relationship of different subareas and suggesting courses of action when possible. View Show abstract Machine Learning Based Auto-Scaling for Containerized Applications Article Full-text available Jul 2020 NEURAL COMPUT APPL Mahmoud Imdoukh Imtiaz Ahmad Mohammad Gh. Alfailakawi Containers are shaping the new era of cloud applications due to their key benefits such as lightweight, very quick to launch, consume minimum resources to run an application which reduces cost, and can be easily and rapidly scaled up/down as per work-load requirements. However, container based cloud applications require sophisticated auto-scaling methods that automatically and in a timely manner provision and de-provision cloud resources without human intervention in response to dynamic fluctuations in work-load. To address this challenge, in this paper, we propose a proactive machine learning based approach to perform auto-scaling of Docker containers in response to dynamic workload changes at runtime. The proposed auto-scaler architecture follows the commonly abstracted four steps of the MAPE (Monitor, Analyze, Plan and Execute) control loop. The monitor component continuously collects different types of data (HTTP request statistics, CPU and memory utilization) that are needed during the analysis and planning phase to determine proper scaling actions. We employ in analysis phase a concise yet fast, adaptive and accurate prediction model based on Long Short-Term Memory (LSTM) neural network to predict future HTTP workload to determine the number of containers needed to handle requests ahead of time to eliminate delays caused by starting or stopping running containers. Moreover, in the planning phase, the proposed Gradually Decreasing Strategy (GDS) avoids oscillations which happens when scal-Address(es) of author(s) should be given ing operations are too frequent. Experimental results using realistic workload show that the prediction accuracy of LSTM model is as accurate as Auto Regression Integrated Moving Average (ARIMA) model but offers 600 times prediction speedup. Moreover, as compared with Artificial Neural Network (ANN) model, LSTM model performs better in terms of auto-scaler metrics related to provisioning and elastic speedup. In addition , it was observed that when LSTM model is used, the predicted workload helped in using the minimum number of replicas to handle future workload. In the experiments, the use of GDS showed promising results in keeping the desired performance at reduced cost to handle cases with sudden workload increase/decrease. View Show abstract Effects of Distance Measure Choice on K-Nearest Neighbor Classifier Performance: A Review Article Full-text available Aug 2019 Haneen Abu Alfeilat Ahmad Hassanat Omar Lasassmeh Surya Prasath The K-nearest neighbor (KNN) classifier is one of the simplest and most common classifiers, yet its performance competes with the most complex classifiers in the literature. The core of this classifier depends mainly on measuring the distance or similarity between the tested examples and the training examples. This raises a major question about which distance measures to be used for the KNN classifier among a large number of distance and similarity measures available? This review attempts to answer this question through evaluating the performance (measured by accuracy, precision, and recall) of the KNN using a large number of distance measures, tested on a number of real-world data sets, with and without adding different levels of noise. The experimental results show that the performance of KNN classifier depends significantly on the distance used, and the results showed large gaps between the performances of different distances. We found that a recently proposed nonconvex distance performed the best when applied on most data sets comparing with the other tested distances. In addition, the performance of the KNN with this top performing distance degraded only ∼20% while the noise level reaches 90%, this is true for most of the distances used as well. This means that the KNN classifier using any of the top 10 distances tolerates noise to a certain degree. Moreover, the results show that some distances are less affected by the added noise comparing with other distances. View Show abstract Simplifying Long Short-Term Memory for Fast Training and Time Series Prediction Article Full-text available Jun 2019 Yuyan Zhang Xin Hao Yong Liu Long short Term Memory(LSTM) has been widely used in sequencial problems. However, for the time series prediction problems, its complex structure limits its running speed and performance. In order to solve this problem, this paper simplified the standard LSTM model by reducing the number of gates and the parameters involved in gates computation. Experiments on univariate data set and multivariate data set show that the proposed simplified model not only has better accuracy, but also has higher running speed. View Show abstract Research on Auto-Scaling of Web Applications in Cloud: Survey, Trends and Future Directions Article Full-text available May 2019 Parminder Singh Pooja Gupta Kiran Jyoti Anand Nayyar Cloud computing emerging environment attracts many applications providers to deploy web applications on cloud data centers. The primary area of attraction is elasticity, which allows to auto-scale the resources on-demand. However, web applications usually have dynamic workload and hard to predict. Cloud service providers and researchers are working to reduce the cost while maintaining the Quality of Service (QoS). One of the key challenges for web application in cloud computing is auto-scaling. The auto-scaling in cloud computing is still in infancy and required detail investigation of taxonomy, approach and types of resources mapped to the current research. In this article, we presented the literature survey for auto-scaling techniques of web applications in cloud computing. This survey supports the research community to find the requirements in auto-scaling techniques. We present a taxonomy of reviewed articles with parameters such as auto-scaling techniques, approach, resources, monitoring tool, experiment, workload, and metric, etc. Based on the analysis, we proposed the new areas of research in this direction. View Show abstract An Open-Source Benchmark Suite for Microservices and Their Hardware-Software Implications for Cloud & Edge Systems Conference Paper Full-text available Apr 2019 Yu Gan Brendon Jackson Kelvin Hu Brian Ritchken Cloud services have recently started undergoing a major shift from monolithic applications, to graphs of hundreds or thousands of loosely-coupled microservices. Microservices fundamentally change a lot of assumptions current cloud systems are designed with, and present both opportunities and challenges when optimizing for quality of service (QoS) and cloud utilization. In this paper we explore the implications microservices have across the cloud system stack. We first present DeathStarBench, a novel, open-source benchmark suite built with microservices that is representative of large end-to-end services, modular and extensible. DeathStarBench includes a social network, a media service, an e-commerce site, a banking system, and IoT applications for coordination control of UAV swarms. We then use DeathStarBench to study the architectural characteristics of microservices, their implications in networking and operating systems, their challenges with respect to cluster management, and their trade-offs in terms of application design and programming frameworks. Finally, we explore the tail at scale effects of microservices in real deployments with hundreds of users, and highlight the increased pressure they put on performance predictability. View Show abstract Auto-scaling VNFs using Machine Learning to Improve QoS and Reduce Cost Conference Paper Full-text available May 2018 Sabidur Rahman Tanjila Ahmed Minh Huynh Biswanath Mukherjee Virtualization of network functions (as virtual routers, virtual firewalls, etc.) enables network owners to efficiently respond to the increasing dynamicity of network services. Virtual Network Functions (VNFs) are easy to deploy, update, monitor, and manage. The number of VNF instances, similar to generic computing resources in cloud, can be easily scaled based on load. Auto-scaling (of resources without human intervention) has been investigated in academia and industry. Prior studies on auto-scaling use measured network traffic load to dynamically react to traffic changes. In this study, we propose a proactive Machine Learning (ML) based approach to perform auto-scaling of VNFs in response to dynamic traffic changes. Our proposed ML classifier learns from past VNF scaling decisions and seasonal/spatial behavior of network traffic load to generate scaling decisions ahead of time. Compared to existing approaches for ML-based auto-scaling, our study explores how the properties (e.g., start-up time) of underlying virtualization technology impacts QoS and cost savings. We consider four different virtualization technologies: Xen and KVM, based on hy-pervisor virtualization, and Docker and LXC, based on container virtualization. Our results show promising accuracy of the ML classifier. We also demonstrate using realistic traffic load traces and optical backbone network that our ML method improves QoS and saves significant cost for network owners as well as leasers. View Show abstract A Survey and Taxonomy of Self-Aware and Self-Adaptive Cloud Autoscaling Systems Article Full-text available Jun 2018 ACM COMPUT SURV Tao Chen Rami Bahsoon Xin Yao Autoscaling system can reconfigure cloud-based services and applications, through various configurations of cloud so ware and provisions of hardware resources, to adapt to the changing environment at runtime. Such a behavior offers the foundation for achieving elasticity in modern cloud computing paradigm. Given the dynamic and uncertain nature of the shared cloud infrastructure, cloud autoscaling system has been engineered as one of the most complex, sophisticated and intelligent artifacts created by human, aiming to achieve self-aware, self-adaptive and dependable runtime scaling. Yet, existing Self-aware and Self-adaptive Cloud Autoscaling System (SSCAS) is not mature to a state that it can be reliably exploited in the cloud. In this article, we survey the state-of-the-art research studies on SSCAS and provide a comprehensive taxonomy for this eld. We present detailed analysis of the results and provide insights on open challenges, as well as the promising directions that are worth investigated in the future work of this area of research. Our survey and taxonomy contribute to the fundamentals of engineering more intelligent autoscaling systems in the cloud. View Show abstract On the Value of Service Demand Estimation for Auto-Scaling Conference Paper Full-text available Feb 2018 André Bauer Johannes Grohmann Nikolas Herbst Samuel Kounev In the context of performance models, service demands are key model parameters capturing the average time individual requests of different workload classes are actively processed. In a system under load, due to measurement interference, service demands normally cannot be measured directly, however, a number of estimation approaches exist based on high-level performance metrics. In this paper, we show that service demands provide significant benefits for implementing modern auto-scalers. Auto-scaling describes the process of dynamically adjusting the number of allocated virtual resources (e.g., virtual machines) in a data center according to the incoming workload. We demonstrate that even a simple auto-scaler that leverages information about service demands significantly outperforms auto-scalers solely based on CPU utilization measurements. This is shown by testing two approaches in three different scenarios. Our results show that the service demand-based autoscaler outperforms the CPU utilization-based one in all scenarios. Our results encourage further research on the application of service demand estimates for resource management in data centers. View Show abstract ENORM: A Framework For Edge NOde Resource Management Article Full-text available Sep 2017 Nan Wang Blesson Varghese Michail Matthaiou Dimitrios Nikolopoulos Current computing techniques using the cloud as a centralised server will become untenable as billions of devices get connected to the Internet. This raises the need for fog computing, which leverages computing at the edge of the network on nodes, such as routers, base stations and switches, along with the cloud. However, to realise fog computing the challenge of managing edge nodes will need to be addressed. This paper is motivated to address the resource management challenge. We develop the first framework to manage edge nodes, namely the Edge NOde Resource Management (ENORM) framework. Mechanisms for provisioning and auto-scaling edge node resources are proposed. The feasibility of the framework is demonstrated on a PokéMonPok´PokéMon Go-like online game use-case. The benefits of using ENORM are observed by reduced application latency between 20%-80% and reduced data transfer and communication frequency between the edge node and the cloud by up to 95%. These results highlight the potential of fog computing for improving the quality of service and experience. View Show abstract Predictive Analytics for Complex IoT Data Streams Article Full-text available Jun 2017 Adnan Akbar Abdullah Khan Francois Carrez Klaus Moessner The requirements of analyzing heterogeneous data streams and detecting complex patterns in near real-time have raised the prospect of Complex Event Processing (CEP) for many internet of things (IoT) applications. Although CEP provides a scalable and distributed solution for analyzing complex data streams on the fly, it is designed for reactive applications as CEP acts on near real-time data and does not exploit historical data. In this regard, we propose a proactive architecture which exploits historical data using machine learning (ML) for prediction in conjunction with CEP. We propose an adaptive prediction algorithm called Adaptive Moving Window Regression (AMWR) for dynamic IoT data and evaluated it using a real-world use case with an accuracy of over 96%. It can perform accurate predictions in near real-time due to reduced complexity and can work along CEP in our architecture. We implemented our proposed architecture using open source components which are optimized for big data applications and validated it on a use-case from Intelligent Transportation Systems (ITS). Our proposed architecture is reliable and can be used across different fields in order to predict complex events. View Show abstract Auto-Scaling Web Applications in Clouds: A Taxonomy and Survey Article Full-text available Sep 2016 ACM COMPUT SURV Chenhao Qu Rodrigo N. Calheiros Rajkumar Buyya Web application providers have been migrating their applications to cloud data centers, attracted by the emerging cloud computing paradigm. One of the appealing features of cloud is elasticity. It allows cloud users to acquire or release computing resources on demand, which enables web application providers to auto-scale the resources provisioned to their applications under dynamic workload in order to minimize resource cost while satisfying Quality of Service (QoS) requirements. In this paper, we comprehensively analyze the challenges remain in auto-scaling web applications in clouds and review the developments in this field. We present a taxonomy of auto-scaling systems according to the identified challenges and key properties. We analyze the surveyed works and map them to the taxonomy to identify the weakness in this field. Moreover, based on the analysis, we propose new future directions. View Show abstract An efficient modelling approach for forecasting financial time series data using support vector regression and windowing operators Article Full-text available Jan 2015 Risul Islam Rasel Nasrin Sultana Phayung Meesad Forecasting or predicting stock market price and trend is regarded as a challenging task because of its chaotic nature. Stock market is essentially a nonlinear, non-parametric, noisy and deterministically chaotic system because of liquid money, stock adequacy, human behaviour, news related to the stock market, gambling, international money rate and so on. Since it is an emerging sector of the business and also many people are related to this sector, many researchers and experts have their interest to work on this area in order to describe this chaotic system for pattern recognition purpose of the trend. Many researches have been already done and still studies are ongoing for better solutions. Artificial intelligence and machine learning are the major techniques that have been applied in different studies. The aim of this study is also using machine learning algorithm for recognising the pattern of stock market trend in order to predict stock price. For that, support vector regression is used as machine learning technique and different widowing operators are used as input selection technique or data pre-processing steps. Experiments are undertaken on three different stock indexes; Dhaka Stock Exchange (DSE), from Bangladesh, S&P 500 stock index and IBM index. View Show abstract On evaluating stream learning algorithms Article Full-text available Oct 2013 João Gama Raquel Sebastião Pedro Pereira Rodrigues Most streaming decision models evolve continuously over time, run in resource-aware environments, and detect and react to changes in the environment generating data. One important issue, not yet convincingly addressed, is the design of experimental work to evaluate and compare decision models that evolve over time. This paper proposes a general framework for assessing predictive stream learning algorithms. We defend the use of prequential error with forgetting mechanisms to provide reliable error estimators. We prove that, in stationary data and for consistent learning algorithms, the holdout estimator, the prequential error and the prequential error estimated over a sliding window or using fading factors, all converge to the Bayes error. The use of prequential error with forgetting mechanisms reveals to be advantageous in assessing performance and in comparing stream learning algorithms. It is also worthwhile to use the proposed methods for hypothesis testing and for change detection. In a set of experiments in drift scenarios, we evaluate the ability of a standard change detection algorithm to detect change using three prequential error estimators. These experiments point out that the use of forgetting mechanisms (sliding windows or fading factors) are required for fast and efficient change detection. In comparison to sliding windows, fading factors are faster and memoryless, both important requirements for streaming applications. Overall, this paper is a contribution to a discussion on best practice for performance assessment when learning is a continuous process, and the decision models are dynamic and evolve over time. View Show abstract An analysis of adaptive windowing for time series forecasting in dynamic environments: Further tests of the DyFor GP model Conference Paper Full-text available Jul 2008 Neal Wagner Zbigniew Michalewicz ABSTRACT Genetic Programming (GP) has proved its applicability for time series forecasting in a number of studies. The Dynamic Forecasting Genetic Program (DyFor GP) model builds on the GP technique by adding features that are tailored for the forecasting of time series whose underlying data-generating processes are non-static. Such time series often appear for real-world forecasting concerns in which environmental con- ditions are constantly changing. In a previous study the Dy- For GP model was shown to improve upon the performance of GP and other benchmark,models for a set of simulated and real time series. The distinctive feature of DyFor GP is its adaptive data window,adjustment. This feedback-driven window,adjustment is designed to automatically hone in on the currently active process in an environment where the View Show abstract The Vision Of Autonomic Computing Article Full-text available Feb 2003 COMPUTER Jeffrey O. Kephart David M. Chess A 2001 IBM manifesto observed that a looming software complexity crisis -caused by applications and environments that number into the tens of millions of lines of code - threatened to halt progress in computing. The manifesto noted the almost impossible difficulty of managing current and planned computing systems, which require integrating several heterogeneous environments into corporate-wide computing systems that extend into the Internet. Autonomic computing, perhaps the most attractive approach to solving this problem, creates systems that can manage themselves when given high-level objectives from administrators. Systems manage themselves according to an administrator's goals. New components integrate as effortlessly as a new cell establishes itself in the human body. These ideas are not science fiction, but elements of the grand challenge to create self-managing computing systems. View Show abstract Horizontal Auto-Scaling for Multi-Access Edge Computing Using Safe Reinforcement Learning Article Nov 2021 ACM T EMBED COMPUT S Kaustabha Ray Ansuman Banerjee Multi-Access Edge Computing (MEC) has emerged as a promising new paradigm allowing low latency access to services deployed on edge servers to avert network latencies often encountered in accessing cloud services. A key component of the MEC environment is an auto-scaling policy which is used to decide the overall management and scaling of container instances corresponding to individual services deployed on MEC servers to cater to traffic fluctuations. In this work, we propose a Safe Reinforcement Learning (RL)-based auto-scaling policy agent that can efficiently adapt to traffic variations to ensure adherence to service specific latency requirements. We model the MEC environment using a Markov Decision Process (MDP). We demonstrate how latency requirements can be formally expressed in Linear Temporal Logic (LTL). The LTL specification acts as a guide to the policy agent to automatically learn auto-scaling decisions that maximize the probability of satisfying the LTL formula. We introduce a quantitative reward mechanism based on the LTL formula to tailor service specific latency requirements. We prove that our reward mechanism ensures convergence of standard Safe-RL approaches. We present experimental results in practical scenarios on a test-bed setup with real-world benchmark applications to show the effectiveness of our approach in comparison to other state-of-the-art methods in literature. Furthermore, we perform extensive simulated experiments to demonstrate the effectiveness of our approach in large scale scenarios. View Show abstract Horizontal Auto-Scaling in Edge Computing Environment using Online Machine Learning Conference Paper Sep 2021 Paulo F. Pires Aluizio Rocha Neto Thiago Pereira da Silva Thaís Vasconcelos Batista View Optimizing Resource Allocation in Edge-Distributed Stream Processing Conference Paper Sep 2021 Aluizio Rocha Neto Thiago Pereira da Silva Thaís Vasconcelos Batista Frederico Lopes Emerging Web applications based on distributed IoT sensor systems and machine intelligence, such as in smart city scenarios, have posed many challenges to network and processing infrastructures. For example, environment monitoring cameras generate massive data streams to event-based applications that require fast processing for immediate actions. Finding a missing person in public spaces is an example of these applications, since his/her location is a piece of perishable information. Recently, the integration of edge computing with machine intelligence has been explored as a promising strategy to interpret such massive data near the sensor and reduce the end-to-end latency of processing events. However, due to the limited capacity and heterogeneity of edge resources, the placement of task processing is not trivial, especially when applications have different quality of service (QoS) requirements. In this paper, we develop an algorithm to solve the optimization problem of allocating a set of nodes with sufficient processing capacity to execute a pipeline of tasks while minimizing the operational cost related to latency and energy and maximizing availability. We compare our algorithm with the resource allocation algorithms (first-fit, best-fit, and worst-fit), achieving a lower cost in scenarios with different nodes’ heterogeneity. We also demonstrate that distributing processing across multiple edge nodes reduces latency and energy consumption and still improves availability compared to processing only in the cloud. View Show abstract Deep Q‐network‐based auto scaling for service in a multi‐access edge computing environment Article Jun 2021 Do-Young Lee Se-Yeon Jeong Kyung‐Chan Ko James Won-Ki Hong In 5G networks, it is necessary to provide services while meeting various service requirements, such as high data rates and low latency, in response to dynamic network conditions. Multi‐access edge computing (MEC) is a promising concept to meet these requirements. The MEC environment enables service providers to deploy their low latency services that are composed of multiple components. However, operating a service manually and attempting to satisfy the quality of service (QoS) requirements is difficult because many factors need to be considered in an MEC scenario. In this paper, we propose an auto‐scaling method using deep Q‐networks (DQN), which is a reinforcement learning algorithm, to resize the number of instances assigned to service. In our evaluation, compared to other baseline methods, the proposed approach maintains the appropriate number of instances effectively in response to dynamic traffic change while satisfying QoS and minimizing the cost of operating the service in the MEC environment. The proposed method was implemented as a module running in OpenStack and published as open‐source software. View Show abstract Efficient Hardware Implementations of Deep Neural Networks: A Survey Conference Paper Jan 2020 Sunny Bodiwala Nirali Nanavati View Resource provisioning for IoT services in the fog computing environment: An autonomic approach Article Jul 2020 COMPUT COMMUN Masoumeh Etemadi Mostafa Ghobaei-Arani Ali Shahidinejad In the recent years, the Internet of Things (IoT) services has been increasingly applied to promote the quality of the human life and this trend is predicted to stretch for into future. With the recent advancements in IoT technology, fog computing is emerging as a distributed computing model to support IoT functionality. Since the IoT services will experience workload fluctuations over time, it is important to automatically provide the proper number of sufficient fog resources to address the workload changes of IoT services to avoid the over- or under-provisioning problems, meeting the QoS requirements at the same time. In this paper, an efficient resource provisioning approach is presented. This approach is inspired by autonomic computing model using Bayesian learning technique to make decisions about the increase and decrease in the dynamic scaling fog resources to accommodate the workload from IoT services in the fog computing environment. Also, we design an autonomous resource provisioning framework based on the generic fog environment three-tier architecture. Finally, we validate the effectiveness of our solution under three workload traces. The simulation results indicate that the proposed solution reduces the total cost and delay violation, and increases the fog node utilization compared with the other methods. View Show abstract Deep Learning Based Resource Allocation For Auto-Scaling VNFs Conference Paper Dec 2019 Yashwant Patel Deepak Verma Rajiv Misra View Convergence of Edge Computing and Deep Learning: A Comprehensive Survey Article Jan 2020 Xiaofei Wang Yiwen Han Victor C. M. Leung Xu Chen Ubiquitous sensors and smart devices from factories and communities are generating massive amounts of data, and ever-increasing computing power is driving the core of computation and services from the cloud to the edge of the network. As an important enabler broadly changing people’s lives, from face recognition to ambitious smart factories and cities, developments of artificial intelligence (especially deep learning, DL) based applications and services are thriving. However, due to efficiency and latency issues, the current cloud computing service architecture hinders the vision of “providing artificial intelligence for every person and every organization at everywhere”. Thus, unleashing DL services using resources at the network edge near the data sources has emerged as a desirable solution. Therefore, edge intelligence, aiming to facilitate the deployment of DL services by edge computing, has received significant attention. In addition, DL, as the representative technique of artificial intelligence, can be integrated into edge computing frameworks to build intelligent edge for dynamic, adaptive edge maintenance and management. With regard to mutually beneficial edge intelligence and intelligent edge, this paper introduces and discusses: 1) the application scenarios of both; 2) the practical implementation methods and enabling technologies, namely DL training and inference in the customized edge computing framework; 3) challenges and future trends of more pervasive and fine-grained intelligence. We believe that by consolidating information scattered across the communication, networking, and DL areas, this survey can help readers to understand the connections between enabling technologies while promoting further discussions on the fusion of edge intelligence and intelligent edge, i.e., Edge DL. View Show abstract Distributing Intelligence to the Edge and Beyond [Research Frontier] Article Nov 2019 IEEE COMPUT INTELL M Edgar Ramos Roberto Morabito Jani-Pekka Kainulainen Machine Intelligence (MI) technologies have revolutionized the design and applications of computational intelligence systems, by introducing remarkable scientific and technological enhancements across domains. MI can improve Internet of Things (IoT) in several ways, such as optimizing the management of large volumes of data or improving automation and transmission in large-scale IoT deployments. When considering MI in the IoT context, MI services deployment must account for the latency demands and network bandwidth requirements. To this extent, moving the intelligence towards the IoT end-device aims to address such requirements and introduces the notion of Distributed MI (D-MI) also in the IoT context. However, current D-MI deployments are limited by the lack of MI interoperability. Currently, the intelligence is tightly bound to the application that exploits it, limiting the provisioning of that specific intelligence service to additional applications. The objective of this article is to propose a novel approach to cope with such constraints. It focuses on decoupling the intelligence from the application by revising the traditional device's stack and introducing an intelligence layer that provides services to the overlying application layer. This paradigm aims to provide final users with more control and accessibility of intelligence services by boosting providers' incentives to develop solutions that could theoretically reach any device. Based on the definition of this emerging paradigm, we explore several aspects related to the intelligence distribution and its impact in the whole MI ecosystem. View Show abstract Auto-scaling Applications in Edge Computing: Taxonomy and Challenges Conference Paper Dec 2017 Salman Taherizadeh Vlado Stankovski The perspective of online services such as Internet of Things (IoT) applications has impressively evolved over the last recent years as they are becoming more and more time-sensitive, maintained at decentralized locations and easily affected by the changing workload intensity at runtime. As a consequence, an up-and-coming trend has been emerging from previously centralized computation to distributed edge computing in order to address these new concerns. The goal of the present paper is therefore twofold. At first, to analyze modern types of edge computing applications and their auto-scaling challenges to offer desirable performance in conditions where the workload dynamically changes. Secondly, to present a new taxonomy of auto-scaling applications. This taxonomy thoroughly considers edge computing paradigm and its complementary technologies such as container-based visualization. View Show abstract Online Learning: A Comprehensive Survey Article Feb 2018 NEUROCOMPUTING Steven C. H. Hoi Doyen Sahoo Jing Lu Peilin Zhao Online learning represents an important family of machine learning algorithms, in which a learner attempts to resolve an online prediction (or any type of decision-making) task by learning a model/hypothesis from a sequence of data instances one at a time. The goal of online learning is to ensure that the online learner would make a sequence of accurate predictions (or correct decisions) given the knowledge of correct answers to previous prediction or learning tasks and possibly additional information. This is in contrast to many traditional batch learning or offline machine learning algorithms that are often designed to train a model in batch from a given collection of training data instances. This survey aims to provide a comprehensive survey of the online machine learning literatures through a systematic review of basic ideas and key principles and a proper categorization of different algorithms and techniques. Generally speaking, according to the learning type and the forms of feedback information, the existing online learning works can be classified into three major categories: (i) supervised online learning where full feedback information is always available, (ii) online learning with limited feedback, and (iii) unsupervised online learning where there is no feedback available. Due to space limitation, the survey will be mainly focused on the first category, but also briefly cover some basics of the other two categories. Finally, we also discuss some open issues and attempt to shed light on potential future research directions in this field. View Show abstract A Lightweight Auto-Scaling Mechanism for Fog Computing in Industrial Applications Article Oct 2018 IEEE T IND INFORM Fan-Hsun Tseng Ming-Shiun Tsai Chia-Wei Tseng Li-Der Chou Fog computing provides a more flexible service environment than cloud computing. The lightweight fog environment is suitable for industrial applications. In order to strengthen service scalability, container virtualization has been proposed and studied in recent years. It is vital to explore the trade-off between service scalability and operating expenses. The paper integrates hypervisor technique with container virtualization, and constructs an integrated virtualization (IV) fog platform for deploying industrial applications based on virtual network function. The paper presents a fuzzy-based real-time auto-scaling (FRAS) mechanism and implements it in the IV fog platform. The FRAS mechanism provides a dynamic, rapid, lightweight and low cost solution to service auto-scaling problem. Experimental results showed that the proposed FRAS mechanism yields a better service scale with lower average delay, error rate and operating expenses compared to other auto-scaling schemes. View Show abstract Modeling and Analyzing MAPE-K Feedback Loops for Self-Adaptation Conference Paper May 2015 Paolo Arcaini Elvinia Riccobene Patrizia Scandurra View An Introduction to Kernel and Nearest-Neighbor Nonparametric Regression Article Aug 1992 N.S. Altman Nonparametric regression is a set of techniques for estimating a regression curve without making strong assumptions about the shape of the true regression function. These techniques are therefore useful for building and checking parametric models, as well as for data description. Kernel and nearest-neighbor regression estimators are local versions of univariate location estimators, and so they can readily be introduced to beginning students and consulting clients who are familiar with such summaries as the sample mean and median. View Show abstract Recommended publications Discover more about: Computing Article Full-text available Resource Provisioning Based Scheduling Framework for Execution of Heterogeneous and Clustered Worklo... September 2019 · Journal of Grid Computing Rajkumar Buyya Sukhpal Singh Gill Provisioning of adequate resources to cloud workloads depends on the Quality of Service (QoS) requirements of these cloud workloads. Based on workload requirements (QoS) of cloud users, discovery and allocation of best workload-resource pair is an optimization problem. Acceptable QoS can be offered only if provisioning of resources is appropriately controlled. So, there is a need for a QoS-based ... [Show full abstract] resource provisioning framework for the autonomic scheduling of resources to observe the behavior of the services and adjust it dynamically in order to satisfy the QoS requirements. In this paper, framework for self-management of cloud resources for execution of clustered workloads named as SCOOTER is proposed that efficiently schedules the provisioned cloud resources and maintains the Service Level Agreement (SLA) by considering properties of self-management and the maximum possible QoS parameters are required to improve cloud based services. Finally, the performance of SCOOTER has been evaluated in a cloud environment that demonstrates the optimized QoS parameters such as execution cost, energy consumption, execution time, SLA violation rate, fault detection rate, intrusion detection rate, resource utilization, resource contention, throughput and waiting time. View full-text Article IDS fitted Q improvement using fuzzy approach for resource provisioning in cloud November 2016 · Journal of Intelligent and Fuzzy Systems Maryam Amiri Mohammad Reza Feizi Derakhshi Leili Mohammad Khanli Reinforcement Learning (RL) is used to find the best policy. A policy is a rule that maps a given state to an appropriate action. The RL is used to learn utility functions for dynamic resource allocation. According to the future demand of resources, the learned policy maps the appropriate resources in a way that wasting energy and resources is stopped and Service Level Agreements (SLA) violation ... [Show full abstract] and Quality of Service (QoS) dropping are also avoided. However, The RL encounters a lot of problems in this field such as: having good policies in the early phases of learning and the learning time to converge to the optimal policy. This paper deals with these problems using the appropriate initialization of Q learning and a new fuzzy approach to increase the convergence speed to the optimal policy. The fuzzy approach presented in this paper improves the accuracy and speed of convergence of Q learning. Firstly, the proposed method predicts the future workload, then determines the appropriate number of physical machines by using the optimal policy learned by improved Q learning. The evaluation results show the advantages of accuracy and convergence speed of the proposed method in comparison with the similar methods. Read more Chapter Quality of Service Support Through a Self-adaptive System in Edge Computing Environments January 2023 Abdullah Fawaz Aljulayfi Karim Djemame Edge Computing (EC) has gained a lot of attention from both industry and academia as it is an essential paradigm that addresses the Quality of Service (QoS) of Internet of Things (IoT) applications. The QoS metrics are defined in a Service Level Agreement (SLA) that must be fulfilled by Service Provider (SP). Indeed, the management of QoS in EC is not a trivial process due to the dynamicity ... [Show full abstract] nature of both EC and IoT devices as well as the IoT applications’ requirements. This rises the need to develop a Self-adaptive System (SAS) that is aware of IoT- and EC-nature and provides a continuous management over the operation environment with consideration to the optimization objectives. Self-adaptation is seen as a promising method to manage such objectives in an efficient manner. In this paper, a QoS framework embedded in a SAS is proposed with respect to the EC environment in terms of workload fluctuation and limited resources. Its design also evaluated using a comprehensive simulation-based investigation that considers the most suitable scheduling algorithm, resource threshold, and resource monitoring interval. The simulation results show that the considered parameters can significantly improve the targeted objectives, which can be up to ~35% and more than 50% in acceptance rate and processing time, respectively. Such improvement is related to understanding the relationship among these parameters and the adopted workload. Read more Looking for the full-text? You can request the full-text of this article directly from the authors on ResearchGate. Request full-text Already a member? Log in ResearchGate iOS App Get it from the App Store now. Install Keep up with your stats and more Access scientific knowledge from anywhere or Discover by subject area Recruit researchers Join for free Login Email Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google Welcome back! Please log in. Email · Hint Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google No account? Sign up Company About us News Careers Support Help Center Business solutions Advertising Recruiting	https://www.researchgate.net/publication/365608335_Online_machine_learning_for_auto-scaling_in_the_edge_computing
Eugene O'Neill - New World Encyclopedia Eugene O'Neill Eugene O'Neill Eugene O'Neill, American playwright Born October 16, 1888 New York, New York Died November 27, 1953 Boston, Massachusetts Eugene Gladstone O'Neill(October 16, 1888 – November 27, 1953) was aNobelandPulitzer Prize-winning American playwright. More than any other dramatist, O'Neill introduced the dramaticRealismpioneered by the European playwrightsAnton Chekhov,Henrik Ibsen, andAugust Strindberginto American theater. O'Neill was also famous for the bleak and tragic tone of his plays, which persistently examine the crushed hopes and dreams of the underprivileged. O'Neill's plays are tragic, but in a new, modern sense of the term. Ancient tragedy depicted noble characters who nonetheless suffered from a tragic flaw. In the realistic theater of O'Neill and other modern dramatists, the characters possess little nobility—they are ordinary people, not of the type of King Oedipus—and are deeply flawed. Yet, for brief moments they are able to transcend their weaknesses to commit acts of "small" heroism and self-sacrifice. With the equalization of society that takes place in modernity, as each individual life becomes more important, less significance is ascribed to the grand hero who represents the whole society (Nietzschebemoaned this democratic attitude). The other strand in O'Neil's plays is a sense of hopeless despair rooted in the loss of meaning in the modern world. Without a clear sense of meaning and purpose, in a world without the certainties ofreligion, his characters engage in the kind of self-destructive behavior that would eventually claim his own life. Contents 1 Life 2 Works 2.1 The Iceman Cometh 2.1.1 Summary 2.1.2 Themes 3 Selected works 4 References 5 External links 6 Credits O'Neill is often considered to be the most influential American playwright of the twentieth century. Just as Ibsen had done decades earlier for the European theater, O'Neill revolutionized the conception of what acceptable drama could be. He was a relentless examiner of the hopes—and failings—of everyday American life, and he sought to present all the aspects of his characters' lives in his plays. As a result of this, O'Neill became notorious for the shocking nature of many of his works; but the overpowering literary merit of his plays holds up past all controversy. LikeHonore de Balzac, O'Neill sought to capture, in his plays, a microcosm of his times. His works reveal the all-to-frequent failures of human life, and in so doing redeems them. Life Eugene O'Neill's life was intimately connected to New London,Connecticut. His father was an Irish-born stage actor named James O'Neill, who had grown up in impoverished circumstances. His mother, Ella Quinlan O'Neill, was the emotionally fragile daughter of a wealthy father who died when she was seventeen. O'Neill's mother never recovered from the death of her second son, Edmund, who had died ofmeaslesat the age of two, and she became addicted to morphine as a result of Eugene's difficult birth. O'Neill was born in a Broadway hotel room. Because of his father's profession, he spent his early years backstage at theaters and on trains as the family moved from place to place. When he was seven, O'Neill was sent to a Catholic boarding school where he found his only solace in books. After being suspended fromPrinceton Universityfor his frequent drinking, O'Neill spent several years as a sailor, during which time he suffered from depression and severe alcoholism. O'Neill lived for six years as a wanderer, working occasionally as a sailor and spending a great deal of time as an unemployed drifter inBuenos Aires, Liverpool, andNew York City. O'Neill would later jokingly refer to this time of his life as his "real education." O'Neill briefly found employment during this period as a writer for the New London Telegraph,dabbling in playwriting from time to time. It wasn't, however, until his experience at Gaylord Farms Sanatorium (where he was recovering from tuberculosis) that he experienced an epiphany and devoted his life to writing plays. O'Neill enrolled in the famous playwriting course taught by George Pierce Baker atHarvard University, spending 1914-15 writing prolifically, though he would later disown all his writings from this period. In 1916, O'Neill had his first big break, when he joined the Provincetown Players, a raggedy band of young writers, artists, and actors who had assembled in the tiny coastal village of Provincetown. Although many other writers wrote plays for the company to perform, O'Neill soon became their biggest attraction. During this period, O'Neill concentrated primarily on writing small, one-act plays that drew heavily from his experiences at sea. Bound East for Cardiffwould become the most famous of these, and it would ultimately be O'Neill's first work to be performed in New York City, to rave reviews. Following the success of Bound East for Cardiff,O'Neill moved back to New York and became a regular on the Greenwich Village literary scene, where he also befriended many radicals, most notably U.S. Communist Party founderJohn Reed. In 1920, O'Neill's first full-length play, Beyond the Horizon,was produced on Broadway. O'Neill would win aPulitzer Prizefor the play, and soon after he became a major literary celebrity. His productivity during this period was legendary; he wrote several plays a year, obsessively revising earlier drafts of plays for reproduction. In 1929, O'Neill moved to the Loire Valley of northwestFrance, where he lived in the Chateau du Plessis in St. Antoine-du-Rocher, Indre-et-Loire. Later, he moved to Danville, California, in 1937, living there until 1944. In 1943, O'Neill disowned his daughter, Oona, for marrying the English actor/director/producerCharlie Chaplinwhen she was 18 and he was 54. He never saw her again. After suffering from multiple health problems (includingalcoholism) over many years, O'Neill ultimately began to suffer from a severe tremor in his hands which made it impossible for him to write. He attempted to write via dictation, but found it impossible to compose by that method; O'Neill never wrote another play for the remaining ten years of his life. O'Neill died from the advanced stages of Parkinson's disease in room 401 of the Shelton Hotel in Boston, on November 27, 1953, at the age of 65. He was interred in the Forest Hills Cemetery in Jamaica Plain, Massachusetts. Works O'Neill's best-known plays include Desire Under the Elms, Strange Interlude(for which he again won the Pulitzer Prize), Mourning Becomes Electra,and his only comedy Ah, Wilderness!a wistful re-imagining of his own youth as he wished it had been. All of his plays tend to be marked by a darkness of tone—even his comic masterpiece Ah, Wilderness!verges dangerously close to becoming a tragedy—and a piercing degree of insight into the inner lives of his beleaguered characters. His late masterpiece The Iceman Cometh,produced in 1946, directly addressed the issues of doubt and religion which had cropped up throughout his oeuvre. Although his written instructions had stipulated that it not be made public until 25 years after his death, in 1956, O'Neill's wife arranged for his autobiographical masterpiece, Long Day's Journey Into Night,to be published, and produced on stage to tremendous critical acclaim; it is now considered to be his finest play. Other posthumously-published works include A Touch of the Poet(1958) and More Stately Mansions(1967). Both A Touch of the Poetand More Stately Mansionswere parts of a planned "dramatic epic" spanning 11 plays that would follow the life and times of a Boston family from the early 1800s to the present day. O'Neill wrote copious notes concerning the direction of the work, but the severe tremor in his hands prevented him from being able to complete anything other than these two fragments. The Iceman Cometh Summary The Iceman Cometh,universally considered one of O'Neill's masterpieces, is set in Harry Hope's decidedly seedy Greenwich Village saloon and rooming house in 1912. The patrons are all dead-end alcoholics who spend every possible moment either drinking or trying to wheedle free drinks from Harry and the bartenders. They also all look forward to the regular visits of one of the bar's most famous patrons, the salesman Theodore Hickman, known to them as Hickey. Every time Hickey finishes a tour of his territory, which is apparently a wide expanse of the East Coast, he typically turns up at the saloon and starts a party. He buys drinks for everyone, regales them with jokes and stories, and goes on a bender of several days until his money runs out, only to start all over again. As the play opens, the regulars are expecting Hickey to turn up soon and plan to throw him a surprise birthday party. The entire first act introduces the various characters, showing them bickering among each other, showing just how drunk and delusional they truly are, all the while awaiting the arrival of Hickey. When Hickey finally arrives, his behavior throws the other characters into turmoil. He appears as charismatic and cheerful as ever, but something has changed; he has sworn himself to sobriety, and he sees life clearly now as never before. He hectors his former drinking companions that they are meaninglessly clinging to dreams of positive change in their lives, while continuing to drown their sorrows exactly as before. Hickey wants the characters to cast away their delusions and embrace the hopelessness of their fates. Themes Following Hickey's return to the saloon, The Iceman Comethconcerns itself explicitly with all of O'Neill favorite themes: Redemption, failure, and fate. The play contains numerous allusions to the Bible, and as the play goes on it becomes clear the Hickey is meant as an allegorical figure for Christ. Yet Hickey, even after his "awakening," is no ordinary savior. Instead of imploring his companions to change their lives and escape the constant cycle of drinking and sorrow, Hickey—with all the fervent zealousness of a real religious prophet—encourages them to simply give in and become content with their own hopelessness. Instead of drinking their lives away, Hickey implores his friends to let go of their sorrows by simply giving up any hope of ever being any happier than they are. Continuing on with this rather ambiguous, almost nihilistic message, the play ends ambiguously, and the audience is left to wonder just how ironic a savior Hickey really is. Selected works Bound East for Cardiff, 1916 The Emperor Jones, 1920 The Hairy Ape, 1922 Anna Christie, 1922 The Fountain, 1923 Marco Millions, 1923-1925 Desire Under the Elms, 1925 Lazarus Laughed, 1925-1926 The Great God Brown, 1926 Strange Interlude, 1928 Dynamo, 1929 Mourning Becomes Electra, 1931 Ah, Wilderness! 1933 Days Without End, 1933 The Iceman Cometh, written 1939, first performed 1946 Long Day's Journey Into Night, written 1941, first performed 1956 A Moon for the Misbegotten, 1943 A Touch of the Poet, completed in 1942, first performed 1958 More Stately Mansions, second draft found in O'Neill's papers, first performed 1967 The Calms of Capricorn, published in 1983 References Black, Stephen A. Eugene O'Neill: Beyond Mourning and Tragedy. Yale University Press, 2002. ISBN 0300093993 . Eugene O'Neill National Historic Site . An Electronic Eugene O'Neill Archive . Nobel autobiography . Eugene O'Neill's Photo & Gravesite . Nobel Prize in Literature : Laureates (1926-1950) 1926: Grazia Deledda \| 1927:Henri Bergson\| 1928: Sigrid Undset \| 1929:Thomas Mann\| 1930:Sinclair Lewis\| 1931: Erik Axel Karlfeldt \| 1932:John Galsworthy\| 1933: Ivan Alekseyevich Bunin \| 1934:Luigi Pirandello\| 1936:Eugene O'Neill\| 1937: Roger Martin du Gard \| 1938:Pearl S. Buck\| 1939: Frans Eemil Sillanpää \| 1944: Johannes Vilhelm Jensen \| 1945:Gabriela Mistral\| 1946:Hermann Hesse\| 1947:André Gide\| 1948:T. S. Eliot\| 1949:William Faulkner\| 1950:Bertrand Russell Complete List Laureates (1901-1925) \| Laureates (1926-1950) \| Laureates (1951-1975) \| Laureates (1976-2000) \| Laureates (2001-2025) Eugene O'Neill history History of "Eugene O'Neill" Categories : Writers and poets	https://www.newworldencyclopedia.org/entry/Eugene_O%27Neill
(PDF) Learning-induced uncertainty reduction in Perceptua decisions is task-dependent PDF \| Perceptual decision-making in which decisions are reached primarily from extracting and evaluating sensory information requires close interactions... \| Find, read and cite all the research you need on ResearchGate May 2014 Frontiers in Human Neuroscience 8:282 DOI: 10.3389/fnhum.2014.00282 Source PubMed License CC BY 3.0 Authors: Feitong Yang Peking University Qiong Wu Florida State University Sheng Li Peking University Download full-text PDF Download full-text PDF Download full-text PDF Read full-text Download citation Copy link Link copied Read full-text Download citation Copy link Link copied Citations (10) References (39) Figures (9) Abstract and Figures Perceptual decision-making in which decisions are reached primarily from extracting and evaluating sensory information requires close interactions between the sensory system and decision-related networks in the brain. Uncertainty pervades every aspect of this process and can be considered related to either the stimulus signal or decision criterion. Here, we investigated the learning-induced reduction of both the signal and criterion uncertainty in two perceptual decision tasks based on two Glass pattern stimulus sets. This was achieved by manipulating spiral angle and signal level of radial and concentric Glass patterns. The behavioral results showed that the participants trained with a task based on criterion comparison improved their categorization accuracy for both tasks, whereas the participants who were trained on a task based on signal detection improved their categorization accuracy only on their trained task. We fitted the behavioral data with a computational model that can dissociate the contribution of the signal and criterion uncertainties. The modeling results indicated that the participants who were trained on the criterion comparison task reduced both the criterion and signal uncertainty. By contrast, the participants who were trained on the signal detection task only reduced their signal uncertainty after training. Our results suggest that the signal uncertainty can be resolved by training participants to extract signals from noisy environments and to discriminate between clear signals, which are evidenced by reduced perception variance after both training procedures. Conversely, the criterion uncertainty can only be resolved by the training of fine discrimination. These findings demonstrate that uncertainty in perceptual decision-making can be reduced with training but that the reduction of different types of uncertainty is task-dependent. \| Stimulus sets for the criterion comparison and signal detection tasks. For the criterion comparison task, the signal level of the stimuli was set to 100%, and the spiral angles varied from 0 • to … \| Model comparison. … \| The categorization accuracy in the test sessions. The performance is shown for (A) the criterion comparison group in the criterion comparison task and the signal detection task, (B) the signal … Stimulus sets for the criterion comparison and signal detection tasks. For the criterion comparison task, the signal level of the stimuli was set to 100%, and the spiral angles varied from 0° to 90°. For the signal detection task, Glass patterns of 0° (radial) and 90° (concentric) spiral angles were presented, and the signal level varied between 0 and 100%. Modified from Li and Yang (2012). … +4 Schematic illustration of the computational model. An example of the Double Model is shown here. The decision process can be considered as comparing the perceived spiral angle of a stimulus (pi) with the implicit decision criterion (ci). If pi > ci, the stimulus was categorized as a concentric pattern. Otherwise, if pi < ci, the stimulus was categorized as a radial pattern. The red curve represents the decision criterion. The two blue curves represent the perceived spiral angle of two stimuli with different signal strengths: the left blue curve represents the perception of a low-signal-strength Glass Pattern whose spiral angle was 30°, and the right blue curve represents the perception of a high-signal-strength Glass Pattern whose spiral angle was 70°. … Figures - uploaded by Feitong Yang Author content All figure content in this area was uploaded by Feitong Yang Content may be subject to copyright. Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations Join for free Public Full-texts 3 fnhum-08-002 82.pdf Content available from CC BY 3.0: fnhum-08-00282.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Content uploaded by Feitong Yang Author content All content in this area was uploaded by Feitong Yang on Aug 31, 2015 Content may be subject to copyright. Learning-induced uncertainty reduction in Perceptua decisions is task-depende nt.pdf Content available from CC BY 3.0: fnhum-08-00282.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Available via license: CC BY 3.0 Content may be subject to copyright. Learning-induced uncertainty reduction in Perceptua decisions is task-depende nt.pdf Content available from CC BY 3.0: fnhum-08-00282.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Learning-induced uncertainty reduction in Perceptua decisions is task-dependent.pdf Available via license: CC BY 3.0 Content may be subject to copyright. A preview of the PDF is not available Citations (10) References (39) ... The previous works reported that the training in the particular task allowed the subjects to improve their behavioral performance. In the visual task, training improved the efficiency of high-level visual processing, which therefore provided less ambiguous sensory information to the decision-related brain networks (Yang et al. (2014) ). While in Ref. (Yang et al. (2014)) the training period has lasted for three days, our results suggest that the training effect is notable even within a 40-min session. ... ... In the visual task, training improved the efficiency of high-level visual processing, which therefore provided less ambiguous sensory information to the decision-related brain networks (Yang et al. (2014)). While in Ref. (Yang et al. (2014) ) the training period has lasted for three days, our results suggest that the training effect is notable even within a 40-min session. The brain's ability to enhance its cognitive performance in the course of training was previously described by Tang and Posner (Tang and Posner (2009)). ... Neuronal adaptation during the prolonged cognitive task improves visual stimulus processing Preprint Full-text available Apr 2020 Vladimir Maksimenko Nikita Frolov Alexander Kuc Mikhail A Lebedev Brain optimally utilizes resources to resist mental fatigue during the prolonged period of cognitive activity. Neural mechanisms underlying long-term cognitive performance remain unknown. We show that during the 40-minutes visual stimuli classification task, subjects improve behavioral performance in terms of response time and correctness. We observe that the prestimulus theta and alpha power grows during the experiment manifesting mental fatigue. The prestimulus beta power, in its turn, increases locally in the region, engaged in the ongoing stimulus processing, that may reflect the neuronal adaptation. Our results evidence that the neuronal adaptation is enhanced in the course of the experiment reducing the cognitive demands required to activate the stimulus-related brain regions. View Show abstract ... In turn, the decreasing RT in the course of the experiment can be associated with the training effect. The role of training in the performance of the perceptual decision-making tasks was demonstrated by Yang et al. (2014) . The authors suggest that the training could improve the efficiency of high-level visual processing, which therefore would provide less ambiguous sensory information to the decision-related brain networks. ... ... The authors suggest that the training could improve the efficiency of high-level visual processing, which therefore would provide less ambiguous sensory information to the decision-related brain networks. While in Yang et al. (2014) the training period lasted for 3 days, our results suggest that the training effect could already be notable even within a 40-min session. ... Neural Interactions in a Spatially-Distributed Cortical Network During Perceptual Decision-Making Article Full-text available Sep 2019 Vladimir Maksimenko Nikita Frolov A. E. Hramov Alexander N Pisarchik Behavioral experiments evidence that attention is not maintained at a constant level, but fluctuates with time. Recent studies associate such fluctuations with dynamics of attention-related cortical networks, however the exact mechanism remains unclear. To address this issue, we consider functional neuronal interactions during the accomplishment of a reaction time (RT) task which requires sustained attention. The participants are subjected to a binary classification of a large number of presented ambiguous visual stimuli with different degrees of ambiguity. Generally, high ambiguity causes high RT and vice versa. However, we demonstrate that RT fluctuates even when the stimulus ambiguity remains unchanged. The analysis of neuronal activity reveals that the subject's behavioral response is preceded by the formation of a distributed functional network in the β-frequency band. This network is characterized by high connectivity in the frontal cortex and supposed to subserve a decision-making process. We show that neither the network structure nor the duration of its formation depend on RT and stimulus ambiguity. In turn, RT is related to the moment of time when the β-band functional network emerges. We hypothesize that RT is affected by the processes preceding the decision-making stage, e.g., encoding visual sensory information and extracting decision-relevant features from raw sensory information. View Show abstract ... This finding is supported by published evidence indicating that decision-making processes are encoded in two separate neural circuits in the brain, in which the DLPFC predominantly affects highly sensitive decisions [15]. This is interesting because, as mentioned in the Introduction, individual differences and task difficulty might have a modulatory effect on response to tDCS intervention and should therefore be carefully considered when interpreting the results [52, 53] . In our study, the referees were classified as interactor referees according to the recent classification provided by Kittel et al. [12]. ... Anodal Transcranial Direct Current Stimulation over the Right Dorsolateral Prefrontal Cortex Boosts Decision Making and Functional Impulsivity in Female Sports Referees Article Full-text available May 2023 Shahrouz Ghayebzadeh Shirin Zardoshtian Ehsan Amiri Daniel Gomes da Silva Machado Abstract: We investigated the effect of anodal transcranial direct current stimulation (tDCS) over the right dorsolateral prefrontal cortex (rDLPFC) on the sensitive decision making of female team sports referees. Twenty-four female referees voluntarily participated in this randomized, double-blind, crossover, and sham-controlled study. In three different sessions, participants received either anodal (a-tDCS; anode (+) over F4, cathode (−) over the supraorbital region (SO)), cathodal (c-tDCS; −F4/+SO), or sham tDCS (sh-tDCS) in a randomized and counterbalanced order. a-tDCS and c-tDCS were applied with 2 mA for 20 min. In sh-tDCS, the current was turned off after 30 s. Before and after tDCS, participants performed the computerized Iowa Gambling Task (IGT) and Go/No Go impul-sivity (IMP) tests. Only a-tDCS improved IGT and IMP scores from pre to post. The delta (Δ = post–pre) analysis showed a significantly higher ΔIGT in a-tDCS compared to c-tDCS (p = 0.02). The ΔIMP was also significantly higher in a-tDCS compared to sh-tDCS (p = 0.01). Finally, the reaction time decreased significantly more in a-tDCS (p = 0.02) and sh-tDCS (p = 0.03) than in c-tDCS. The results suggest that the a-tDCS improved factors related to sensitive decision making in female team sports referees. a-tDCS might be used as an ergogenic aid to enhance decision performance in female team sports referees. View Show abstract ... Moreover, the DDM is certainly not the only model capable of explaining our data. There is a large class of alternative models, with and without temporal integration (these strategies are extremely difficult to differentiate), that could predict the RT distributions (Cartwright and Festinger, 1943;Watson, 1979;Cook and Maunsell, 2002;Ditterich, 2006;Uchida et al., 2006;Trimmer et al., 2008;Yang et al., 2008;Thura et al., 2012; Yang et al., 2014; Latimer et al., 2015;Stine et al., 2020). We must emphasize, however, that our aim was not to find the 'best' predicting tool from a large set of models. ... Response Time Distributions and the Accumulation of Visual Evidence in Freely Moving Mice Article Full-text available Aug 2022 NEUROSCIENCE Mario Treviño Villegas Ricardo Cossyleon Elí Lezama Response time (RT) distributions are histograms of the observed RTs for discriminative choices, comprising a rich source of empirical information to study perceptual processes. The drift-diffusion model (DDM), a mathematical formulation predicting decision tasks, reproduces the RT distributions, contributing to our understanding of these processes from a theoretical perspective. Notably, although the mouse is a popular model system for studying brain function and behavior, little is known about mouse perceptual RT distributions, and their description from an information-accumulation perspective. We combined an automated visual discrimination task with pharmacological micro-infusions of targeted brain regions to acquire thousands of responses from freely-moving adult mice. Both choices and escape latencies showed a strong dependency on stimulus discriminability. By applying a DDM fit to our experimental data, we found that the rate of incoming evidence (drift rate) increased with stimulus contrast but was reversibly impaired when inactivating the primary visual cortex (V1). Other brain regions involved in the decision-making process, the posterior parietal cortex (PPC) and the frontal orienting fields (FOF), also influenced relevant parameters from the DDM. The large number of empirical observations that we collected for this study allowed us to achieve accurate convergence for the model fit. Therefore, changes in the experimental conditions were mirrored by changes in model parameters, suggesting the participation of relevant brain areas in the decision-making process. This approach could help interpret future studies involving attention, discrimination, and learning in adult mice. View Show abstract ... Second, an outstanding difference between our study and these two studies was the task used for training. Previous literature has shown that learning to detect stimuli from a noisy background (e.g., orientation detection task) utilized a different mechanism from learning a fine discrimination task (e.g., vernier task and orientation discrimination task; Chang et al., 2014;Dosher & Lu, 2005; Yang et al., 2014) . The different mechanisms of the two types of tasks could contribute to different levels of competition between neuronal populations recruited by two trainings. ... Simple contextual cueing prevents retroactive interference in short-term perceptual training of orientation detection tasks Article Jun 2022 Hui Huang Yangming Zhang Sheng Li Perceptual training of multiple tasks suffers from interference between the trained tasks. Here, we conducted five psychophysical experiments with separate groups of participants to investigate the possibility of preventing the interference in short-term perceptual training. We trained the participants to detect two orientations of Gabor stimuli in two adjacent days at the same retinal location and examined the interference of training effects between the two orientations. The results showed significant retroactive interference from the second orientation to the first orientation (Experiment 1 and Experiment 2). Introducing a 6-h interval between the pre-test and training of the second orientation did not eliminate the interference effect, excluding the interpretation of disrupted reconsolidation as the pre-test of the second orientation may reactivate and destabilize the representation of the first orientation (Experiment 3). Finally, the training of the two orientations was accompanied by fixations in two colors, each serving as a contextual cue for one orientation. The results showed that the retroactive interference was not evident if the participants passively perceived contextual cues during the training and test sessions (Experiment 4). Importantly, this facilitation effect could be observed if the contextual cues appeared only during the training, demonstrating the robustness of the effect (Experiment 5). Our findings suggest that the retroactive interference effect in short-term perceptual training of orientation detection tasks was likely the result of higher-level factors such as shared contextual cues embedded in the tasks. The efficiency of multiple perceptual trainings could be facilitated by associating the trained tasks with different contextual cues. View Show abstract ... Следует также отметить, что состояние испытуемых может изменяться в процессе прохождения эксперимента. В частности, на восприятие будет влиять когнитивное утомление (mental fatigue) [23], а также эффект тренировки [24] , вызванный продолжительным выполнением схожих задач. В целях минимизации этих воздействий визуальные стимулы для двух рассматриваемых наборов были выбраны в случайном порядке и моменты их предъявлений были равномерно распределены в течение всего эксперимента. ... Study of sensory information processing depending on visual stimulus complexity based on multichannel EEG signalsИсследование процесса обработки зрительной информации в зависимости от ориентации стимулов по многоканальным записям ЭЭГ Article Feb 2020 Vladimir Maksimenko Alexander Kuc Marina Victorovna Khramova A. E. Hramov Introduction: Analysis of electrical activity in the cortical neural network during the processing of visual information is one ofthe most interesting issues in modern neuroscience. The particular attention of the researchers is attracted by the study of neuralactivity during complex visual stimuli processing. Purpose: Studying the process of sensory information processing in the corticalneural network based on recorded electrical activity signals (EEG). Results: We have studied neural activity during visual informationprocessing based on the stimulus-related change in the spectral EEG energy in the 15–30 Hz frequency band. Using the developedapproach, we analyzed the influence of the visual stimulus complexity on the features of spatio-temporal neural activity. It has beenfound that at low complexity the spectral amplitude of the EEG in the range of 15–30 Hz increases mainly in the parietal zone. Withincreasing complexity, the spectral amplitude of the EEG increases simultaneously in different parts of the cortex, mainly in the frontalregion. Practical relevance: The identified features of neural dynamics can be used in the development of passive brain-computerinterfaces to monitor a person’s cognitive state and evaluate the cognitive load in real time. View Show abstract ... A novel feature of this study is that we tested classification of a number of stimuli varying in intensity and assigned to two categories. Most signal detection paradigms test only one (usually low intensity) stimulus representing the signal against signal absent trials (for exceptions, see Kepecs et al., 2008; Yang et al., 2014 ). Interoceptive categories, however, usually subsume a range of different signals (e.g., signs of airway obstruction in asthma may come in different degrees of intensity and a variety of symptoms on multiple dimensions may fall in the category 'cold symptoms') and an important task is to classify ambiguous signals as belonging to one of various sensation classes. ... Accuracy and bias in retrospective symptom reporting Article Jul 2016 Omer Van den Bergh Marta Walentynowicz Purpose of review: Self-reported bodily symptoms are of primary importance in healthcare and in health-related research. Typically, they are assessed in clinical interviews or by means of traditional questionnaire formats that require the respondent to provide retrospective symptom estimates rated along intuitive frequency and/or intensity standards and aggregated across varying or unspecified time windows. Recent findings: Retrospective symptom assessments are often biased when compared to (averaged) momentary assessments of symptoms. A variety of factors and conditions have been identified to influence the amount of bias in symptom reporting. Recent research has focused on the underlying mechanisms for the discrepancy between memory and experience. It is suggested that different types of questions and formats assess different types of information, and each may be relevant for different purposes. Knowledge of these underlying mechanisms also provides a relevant framework to better understand individual differences in symptom reporting, including somatoform and somatic symptom disorder. Summary: Accuracy of self-reported bodily symptoms is important for the clinician and the researcher. Understanding the mechanisms underlying bias may provide an interesting window to understand how symptom episodes are processed, encoded, and consolidated in memory and may also provide clues to modify symptom experiences. View Show abstract ... A novel feature of this study is that we tested classification of a number of stimuli varying in intensity and assigned to two categories. Most signal detection paradigms test only one (usually low intensity) stimulus representing the signal against signal absent trials (for exceptions, see Kepecs et al., 2008; Yang et al., 2014) . Interoceptive categories, however, usually subsume a range of different signals (e.g., signs of airway obstruction in asthma may come in different degrees of intensity and a variety of symptoms on multiple dimensions may fall in the category 'cold symptoms') and an important task is to classify ambiguous signals as belonging to one of various sensation classes. ... Interoception and symptom reporting: Disentangling accuracy and bias Article Full-text available Jun 2015 Sibylle Petersen Ken Van Staeyen Claus Vögele Omer Van den Bergh Anxiety and anxiety sensitivity are positively related to accuracy in the perception of bodily sensations. At the same time, research consistently reports that these traits are positively related to bias, resulting in the report of more and more intense symptoms that poorly correspond with physiological dysfunction. The aim of this study was to test the relationship of accuracy and bias in interoception. Furthermore, we tested the impact of individual differences in negative affect and symptom report in daily life on interoceptive accuracy and bias. Individuals higher in symptom report in daily life and negative affect were marginally more accurate in an interoceptive classification task in which participants were asked to identify different respiratory stimuli (inducing breathing effort) as belonging to a high or low intensity category. At the same time, bias in overestimating intensity of stimuli was significantly increased in participants higher in symptom report and negative affect, but only for more ambiguous stimuli. Results illustrate that interoceptive accuracy and bias need to be considered independently to understand their interaction with psychological factors and to disentangle (mis)perception of bodily sensations from liberal or conservative perceptual decision strategies. View Show abstract Criterion learning-A neglected aspect of training in cytopathology? Article Jul 2018 Andrew Evered Cytologists search for rare signals (neoplastic cells) in visually noisy samples. The difficult nature of the task means that cytologists can never be completely certain about the diagnostic decisions they make. For most visual encounters a cytologist will adopt a probabilistic internal response (e.g. “I am 75% certain that this is an abnormal cell”) but to be effective they must adopt a threshold, or decision criterion, that generates an explicit binary response. In other words, they must learn to decide whether each encountered cell is normal or abnormal. Without this ability cytologists are paralysed. The task is summarized in figure 1, which shows hypothetical frequency distribution curves for responses to normal and abnormal cell populations in a given cytology specimen. This article is protected by copyright. All rights reserved. View Show abstract Religion and Art: A Cognitive and Evolutionary Approach Article Full-text available Feb 2015 Stewart Guthrie Religion and art both have resisted scientific analysis and even definition. Multi-disciplinary research on these two and on cognition, however, supports particular definitions and analyses of both terms by setting them in a context of evolved strategies for dealing with cognitive uncertainty. In my view, what religion and art most importantly share is their attempt (shared also by science) to make sense of the world and their Pascal's Wager-like strategy (nominally not shared by science) of doing so first by positing human characteristics in nonhuman phenomena. This necessary, better-safe-than-sorry strategy cross-culturally results in representing the world as humanlike. View Show abstract Show more Unreliable Evidence: 2 Sources of Uncertainty During Perceptual Choice Article Full-text available Oct 2013 CEREB CORTEX Elizabeth Michael Vincent de Gardelle Alejo Nevado-Holgado Christopher Summerfield Perceptual decisions often involve integrating evidence from multiple concurrently available sources. Uncertainty arises when the integrated (mean) evidence fails to support one alternative over another. However, evidence heterogeneity (variability) also provokes uncertainty. Here, we asked whether these 2 sources of uncertainty have independent behavioral and neural effects during choice. Human observers undergoing functional neuroimaging judged the average color or shape of a multielement array. The mean and variance of the feature values exerted independent influences on behavior and brain activity. Surprisingly, BOLD signals in the dorsomedial prefrontal cortex (dmPFC) showed polar opposite responses to the 2 sources of uncertainty, with the strongest response to ambiguous tallies of evidence (high mean uncertainty) and to homogenous arrays (low variance uncertainty). These findings present a challenge for models that emphasize the role of the dmPFC in detecting conflict, errors, or surprise. We suggest an alternative explanation, whereby evidence is processed with increased gain near the category boundary. View Show abstract Learning Acts on Distinct Processes for Visual Form Perception in the Human Brain Article Full-text available Jan 2012 J NEUROSCI Stephen D Mayhew Sheng Li Zoe Kourtzi Learning is known to facilitate our ability to detect targets in clutter and optimize brain processes for successful visual recognition. Previous brain-imaging studies have focused on identifying spatial patterns (i.e., brain areas) that change with learning, implicating occipitotemporal and frontoparietal areas. However, little is known about the interactions within this network that mediate learning-dependent improvement in complex perceptual tasks (i.e., discrimination of visual forms in clutter). Here we take advantage of the complementary high spatial and temporal resolution of simultaneous EEG-fMRI to identify the learning-dependent changes in spatiotemporal brain patterns that mediate enhanced behavioral sensitivity in the discrimination of global forms after training. We measured the observers' choices when discriminating between concentric and radial patterns presented in noise before and after training. Similarly, we measured the choices of a pattern classifier when predicting each stimulus from EEG-fMRI signals. By comparing the performance of human observers and classifiers, we demonstrated that learning alters sensitivity to visual forms and EEG-fMRI activation patterns related to distinct visual recognition processes. In particular, behavioral improvement after training was associated with changes in (1) early processes involved in the integration of global forms in higher occipitotemporal and parietal areas, and (2) later processes related to categorical judgments in frontal circuits. Thus, our findings provide evidence that learning acts on distinct visual recognition processes and shapes feedforward interactions across brain areas to support performance in complex perceptual tasks. View Show abstract Learning Shapes Spatiotemporal Brain Patterns for Flexible Categorical Decisions Article Full-text available Nov 2011 CEREB CORTEX Sheng Li Stephen D Mayhew Zoe Kourtzi Learning is thought to facilitate our ability to perform complex perceptual tasks and optimize brain circuits involved in decision making. However, little is known about the experience-dependent mechanisms in the human brain that support our ability to make fine categorical judgments. Previous work has focused on identifying spatial brain patterns (i.e., areas) that change with learning. Here, we take advantage of the complementary high spatial and temporal resolution of simultaneous electroencephalography-functional magnetic resonance imaging (EEG-fMRI) to identify the spatiotemporal dynamics between cortical networks involved in flexible category learning. Observers were trained to use different decision criteria (i.e., category boundaries) when making fine categorical judgments on morphed stimuli (i.e., radial vs. concentric patterns). Our findings demonstrate that learning acts on a feedback-based circuit that supports fine categorical judgments. Experience-dependent changes in the behavioral decision criterion were associated with changes in later perceptual processes engaging higher occipitotemporal and frontoparietal circuits. In contrast, category learning did not modulate early processes in a medial frontotemporal network that are thought to support the coarse interpretation of visual scenes. These findings provide evidence that learning flexible criteria for fine categorical judgments acts on distinct spatiotemporal brain circuits and shapes the readout of sensory signals that provide evidence for categorical decisions. View Show abstract Robust averaging during perceptual judgment Article Full-text available Aug 2011 P NATL ACAD SCI USA Vincent de Gardelle Christopher Summerfield An optimal agent will base judgments on the strength and reliability of decision-relevant evidence. However, previous investigations of the computational mechanisms of perceptual judgments have focused on integration of the evidence mean (i.e., strength), and overlooked the contribution of evidence variance (i.e., reliability). Here, using a multielement averaging task, we show that human observers process heterogeneous decision-relevant evidence more slowly and less accurately, even when signal strength, signal-to-noise ratio, category uncertainty, and low-level perceptual variability are controlled for. Moreover, observers tend to exclude or downweight extreme samples of perceptual evidence, as a statistician might exclude an outlying data point. These phenomena are captured by a probabilistic optimal model in which observers integrate the log odds of each choice option. Robust averaging may have evolved to mitigate the influence of untrustworthy evidence in perceptual judgments. View Show abstract Advances in visual perceptual learning and plasticity. Nature Reviews Neuroscience, 11, 53-60 Article Full-text available Dec 2009 Yuka Sasaki José E Náñez Takeo Watanabe Visual perceptual learning (VPL) is defined as a long-term improvement in performance on a visual task. In recent years, the idea that conscious effort is necessary for VPL to occur has been challenged by research suggesting the involvement of more implicit processing mechanisms, such as reinforcement-driven processing and consolidation. In addition, we have learnt much about the neural substrates of VPL and it has become evident that changes in visual areas and regions beyond the visual cortex can take place during VPL. View Show abstract Neural Basis of a Perceptual Decision in the Parietal Cortex (Area LIP) of the Rhesus Monkey Article Oct 2001 Michael N Shadlen William T. Newsome We recorded the activity of single neurons in the posterior parietal cortex (area LIP) of two rhesus monkeys while they discriminated the direction of motion in random-dot visual stimuli. The visual task was similar to a motion discrimination task that has been used in previous investigations of motion-sensitive regions of the extrastriate cortex. The monkeys were trained to decide whether the direction of motion was toward one of two choice targets that appeared on either side of the random-dot stimulus. At the end of the trial, the monkeys reported their direction judgment by making an eye movement to the appropriate target. We studied neurons in LIP that exhibited spatially selective persistent activity during delayed saccadic eye movement tasks. These neurons are thought to carry high-level signals appropriate for identifying salient visual targets and for guiding saccadic eye movements. We arranged the motion discrimination task so that one of the choice targets was in the LIP neuron's response field (RF) while the other target was positioned well away from the RF. During motion viewing, neurons in LIP altered their firing rate in a manner that predicted the saccadic eye movement that the monkey would make at the end of the trial. The activity thus predicted the monkey's judgment of motion direction. This predictive activity began early in the motion-viewing period and became increasingly reliable as the monkey viewed the random-dot motion. The neural activity predicted the monkey's direction judgment on both easy and difficult trials (strong and weak motion), whether or not the judgment was correct. In addition, the timing and magnitude of the response was affected by the strength of the motion signal in the stimulus. When the direction of motion was toward the RF, stronger motion led to larger neural responses earlier in the motion-viewing period. When motion was away from the RF, stronger motion led to greater suppression of ongoing activity. Thus the activity of single neurons in area LIP reflects both the direction of an impending gaze shift and the quality of the sensory information that instructs such a response. The time course of the neural response suggests that LIP accumulates sensory signals relevant to the selection of a target for an eye movement. View Show abstract A Role for Neural Integrators in Perceptual Decision Making Article Nov 2003 CEREB CORTEX Mark E Mazurek Decisions based on uncertain information may benefit from an accumulation of information over time. We asked whether such an accumulation process may underlie decisions about the direction of motion in a random dot kinetogram. To address this question we developed a computational model of the decision process using ensembles of neurons whose spiking activity mimics neurons recorded in the extrastriate visual cortex (area MT or V5) and a sensorimotor association area of the parietal lobe (area LIP). The model instantiates the hypothesis that neurons in sensorimotor association areas compute the time integral of sensory signals from the visual cortex, construed as evidence for or against a proposition, and that the decision is made when the integrated evidence reaches a threshold. The model explains a variety of behavioral and physiological measurements obtained from monkeys. View Show abstract Linear Separability in Classification Learning Article Sep 1981 J Exp Psychol Hum Learn Mem Doug L Medin Paula J. Schwanenflugel Performed 4 experiments to determine whether linearly separable (LS) categories (which can be perfectly partitioned on the basis of a weighted, additive combination of component information) are easier to learn than non-LS categories. 224 Ss, predominantly undergraduates, participated. Independent cue models (e.g., prototype theories) predict that, with average between-category similarity held constant, LS categories will be easier to master. Relational coding models (e.g., the context model) imply that individual cases of high similarity of exemplars to exemplars in contrasting categories are the major determinant of task difficulty. These experiments varied stimulus type (geometric shapes or photographs of faces), category size (small or infinitely large), and instructions. With average similarity controlled, instances of high similarity of exemplars to exemplars influenced performance, but there was no evidence that LS categories were learned more readily than non-LS categories. (29 ref) (PsycINFO Database Record (c) 2012 APA, all rights reserved) View Show abstract Task-dependent uncertainty modulation of perceptual decisions in the human brain Article Oct 2012 EUR J NEUROSCI Sheng Li Feitong Yang Perceptual judgments are frequently made during uncertain situations. Previous human brain imaging studies have revealed multiple cortical and subcortical areas that are activated when decision uncertainty is linked to outcome probability. However, the neural mechanisms of uncertainty modulation in different perceptual decision tasks have not been systematically investigated. Uncertainty of perceptual decision can originate either from highly similar object categories (e.g. tasks based on criterion comparison) or from noise being added to visual stimuli (e.g. tasks based on signal detection). In this study, we used functional magnetic resonance imaging (fMRI) to investigate the neural mechanisms of task-dependent modulation of uncertainty in the human brain during perceptual judgements. We observed correlations between uncertainty levels and fMRI activity in a network of areas responsible for performance monitoring and sensory evidence comparison in both tasks. These areas are associated with late stages of perceptual decision, and include the posterior medial frontal cortex, dorsal lateral prefrontal cortex, and intraparietal sulcus. When the modulation of uncertainty on the two tasks was compared, dissociable cortical networks were identified. Uncertainty in the criterion comparison task modulated activity in the left lateral prefrontal cortex related to rule retrieval. In the signal detection task, uncertainty modulated activity in higher visual processing areas thought to be sensory information 'accumulators' that are active during early stages of perceptual decision. These findings offer insights into the mechanism of information processing during perceptual decision-making. View Show abstract Assessing the Neural Basis of Uncertainty in Perceptual Category Learning through Varying Levels of Distortion Article Jul 2011 J COGNITIVE NEUROSCI Reka Daniel Gerd Wagner Kathrin Koch Ralf Schlösser The formation of new perceptual categories involves learning to extract that information from a wide range of often noisy sensory inputs, which is critical for selecting between a limited number of responses. To identify brain regions involved in visual classification learning under noisy conditions, we developed a task on the basis of the classical dot pattern prototype distortion task [M. I. Posner, Journal of Experimental Psychology, 68, 113-118, 1964]. Twenty-seven healthy young adults were required to assign distorted patterns of dots into one of two categories, each defined by its prototype. Categorization uncertainty was modulated parametrically by means of Shannon's entropy formula and set to the levels of 3, 7, and 8.5 bits/dot within subsets of the stimuli. Feedback was presented after each trial, and two parallel versions of the task were developed to contrast practiced and unpracticed performance within a single session. Using event-related fMRI, areas showing increasing activation with categorization uncertainty and decreasing activation with training were identified. Both networks largely overlapped and included areas involved in visuospatial processing (inferior temporal and posterior parietal areas), areas involved in cognitive processes requiring a high amount of cognitive control (posterior medial wall), and a cortico-striatal-thalamic loop through the body of the caudate nucleus. Activity in the medial prefrontal wall was increased when subjects received negative as compared with positive feedback, providing further evidence for its important role in mediating the error signal. This study characterizes the cortico-striatal network underlying the classification of distorted visual patterns that is directly related to decision uncertainty. View Show abstract Show more Recommendations Discover more Article Full-text available Semantic Packing: An Account for Category Coherence Shohei Hidaka Jun Saiki Linda B Smith It is argued by a computational model that intuitive groupings for natural categories exist, called category coherence. The relationship between property and gen-eralization of category, referred to as smoothness, is pro-posed as a quantitative measure of category coherence. A survey on early acquired noun categories suggests that a basic-level category has smoothness. Similarity-based ... [Show full abstract] categorization models can not account for category co-herence, because the definition of category and feature selection involves an essential circularity. We propose here a semantic packing theory that produces smooth-ness in categories. In the model, the learner's two con-flicting constraints on discrimination and generalization are optimized simultaneously, as if packing knowledge into memory. The equivalence between packing and smoothness is mathematically proven under a specific simplification. Furthermore, the simulation showed that the packing constraint could reproduce the original cat-egory organization based on partial survey data. These results indicate that semantic packing could be a core mechanism of fast mapping, a process by which children generalize a novel instance without the need for trial and error. View full-text Article Old/new differences in direct and indirect memory tests using pictures and words in within- and cros... July 1997 · Cognitive Brain Research Victoria A Kazmerski David Friedman Indirect measures of repetition priming are more sensitive to changes in surface features than are direct measures of memory. This dissociation may reflect differences in the extent to which the two tasks rely on form-specific processes, or on the activation of different memory systems. To assess this, subjects at study made semantic discriminations to a mixed list of pictures and words. At test, ... [Show full abstract] half the concepts were repeated in the surface form presented at study while half were repeated in the other surface form. Subjects in the indirect test continued making the same discrimination, whereas those in the direct test performed a yes/no recognition task. For both tasks, significant old/new within-form differences were found for event-related potential (ERP) and reaction time (RT) measures. Cross-form old/new differences were reliable only for the word-picture condition in the direct task and only for the ERP indices. These data suggest that both direct and indirect memory tasks are influenced by form-specific as well as form-non-specific processing, and that neither the transfer-appropriate processing nor memory systems approaches can completely account for this pattern of results. © 1997 Elsevier Science B.V. All rights reserved. Read more Poster The importance of color and spatial frequency information after laboratory-trained perceptual expert... May 2016 · Journal of Vision Hillary Hadley Erik Arnold Andrea M. Cataldo [...] Lisa S Scott ABSTRACT: Previous expertise training with birds (Scott et al., 2006) and cars (Scott et al., 2008) suggests that subordinate-level training improves perceptual discrimination and increases occipital-temporal event-related potentials (ERPs). Here, we examined whether manipulating color or spatial frequency information influenced discrimination or ERP components after training. Adults completed ... [Show full abstract] 6-10 hours of training with full color images of two sets of objects trained at either the basic or subordinate level. Before and after training, participants completed a subordinate-level sequential matching task while behavioral and ERP responses were recorded. Images were either full color, grayscale, high spatial frequency (HSF: >8 cpd), or low spatial frequency (LSF: <8 cpd) filtered. Analyses of d’ suggest that training increases accuracy following subordinate- but not basic-level training for all image manipulations. Additionally, d’ varied across manipulations, where discrimination was superior for color images, followed by grayscale and HSF-filtered images, and finally LSF-filtered images. Although no training-specific ERP results were found, P1, N170, and N250 component amplitudes differed in response to frequency-filtered images. The current findings are consistent with previous training results (Scott et al., 2006; 2008) and suggest that subordinate-level training increased accuracy in all conditions, despite the loss of color or spatial frequency information. Although not related to training, spatial frequency filtering selectively decreased the P1 amplitude and increased the N170 and N250 amplitudes to degraded images, which may reflect disruption of early object processing. Read more Chapter Novelty Dependent Categorization and Learning in Calm Modules January 1990 Jacob M.J. Murre R. Hans Phaf Gezinus Wolters CALM (Categorizing And Learning Module) forms a basic unit for the construction of multi-modular networks suited for supervised and unsupervised learning. Its design is guided by considerations from a practical, neurobiological, and psychological nature. In CALM networks, modules may or may not be interconnected. If modules are linked, interconnections exist which are modifiable according to a ... [Show full abstract] modified Hebb rule. Bidirectional (nonsymmetric) connections are possible. Categorization and learning speed in a CALM module are dependent on the novelty of a local activation pattern. Single module simulations indicate that module convergence and pattern discrimination of new inputs are learned relatively fast. A CALM module can both discriminate and generalize, depending on module size and structure of the pattern set. CALM networks have, among other things, been applied to pattern (character) recognition tasks, and to the modeling of psychological experiments on the dissociation of explicit and implicit memory. For the construction and evaluation of CALM networks a high level tool has been developed. CALM is currently also being implemented in a 400 processor parallel machine. Read more Last Updated: 09 Mar 2023 Discover the world's research Join ResearchGate to find the people and research you need to help your work. Join for free ResearchGate iOS App Get it from the App Store now. Install Keep up with your stats and more Access scientific knowledge from anywhere or Discover by subject area Recruit researchers Join for free Login Email Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google Welcome back! Please log in. Email · Hint Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google No account? Sign up Company About us News Careers Support Help Center Business solutions Advertising Recruiting	https://www.researchgate.net/publication/262532655_Learning-induced_uncertainty_reduction_in_Perceptua_decisions_is_task-dependent
Full text of District Notices (Federal Reserve Bank of Dallas) : New Offering - Treasury Bills, Circular No. 70-134 \| FRASER \| St. Louis Fed Full text of District Notices (Federal Reserve Bank of Dallas) : New Offering - Treasury Bills, Circular No. 70-134 F ederal reserve Bank of Dallas F I S C A L A G E N T O F T H E U N IT E D S T A T E S DALLAS, TEXAS 75222 Circular No. 70-134 Ju n e 3, 1970 NEW OFFERING — TREASURY BILLS To All Banking Institutions an d Others Concerned in the Eleventh Federal Reserve District: Your attention is invited to the following statement giving details of two issues of Treasury bills: T he T reasury D epartm ent, by this public notice, invites tenders for two series of Treasury bills to the aggregate am ount of $3,100,000,000, or thereabouts, for cash and in exchange for T reasury bills m aturing June 11, 1970, in the am ount of $2,998,363,000, as follows: 91-DAY B IL L S (to m aturity date) to be issued Jun e 11, 1970, in the am ount of $1,800,000,000, or thereabouts, representing an additional am ount of bills dated M arch 12, 1970, and to m ature Septem ber 10, 1970, originally issued in the am ount of $1,301,270,000, the additional and original bills to be freely interchangeable. 182-DAY B IL L S for $1,300,000,000, or thereabouts, to be dated June 11, 1970, and to m ature D ecem ber 10, 1970. T he bills of both series will be issued on a discount basis under com petitive and noncom petitive bidding as hereinafter provided, and a t m aturity their face am ount will be payable w ithout interest. T hey will be issued in bearer form only, and in denom inations of $10,000, $50,000, $100,000, $500,000 and $1,000,000 (m aturity value). T enders will be received a t F ederal Reserve Banks and Branches up to the closing hour, one-thirty p.m., E astern D aylight Saving Tim e, Monday, Jun e 8, 1970. T enders will not be received a t the T reasury D epartm ent, W ashington. Each tender m ust be for an even m ultiple of $10,000, and in the case of com petitive tenders the price offered m ust be expressed on the basis of 100, with not more than three decimals, e.g., 99.925. Fractions m ay not be used. It is urged th a t tenders be made on the printed forms and forwarded in the special envelopes which will be supplied by Federal R eserve Banks or Branches on application therefor. B anking institutions generally m ay subm it tenders for account of customers provided th e names of the customers are set forth in such tenders. Others than banking institutions will not be perm itted to subm it tenders except for their own account. T enders will be received w ithout deposit from incorporated banks and trust companies and from responsible and recognized dealers in investm ent securities. Tenders from others m ust be accompanied by paym ent of 2 percent of the face am ount of Treasury bills applied for, unless the tenders are accom panied by an express guaranty of paym ent by an incorporated bank or tru st company. Im m ediately after the closing hour, tenders will be opened a t the Federal Reserve Banks and Branches, following which public announcem ent will be made by the T reasury D epartm ent of the am ount and price range of accepted bids. Only those subm itting com petitive tenders will be advised of the acceptance or rejection thereof. T he Secretary of the T reasury expressly reserves the right to accept or reject any or all tenders, in whole or in part, and his action in any such respect shall be final. Sub ject to these reservations, noncom petitive tenders for each issue for $200,000 or less w ithout stated price from any one bidder will be accepted in full a t the average price (in three decimals) of accepted com petitive bids for the respective issues. S ettlem ent for accepted tenders in accordance with the bids m ust be m ade or com pleted at the F ederal Reserve B ank on Ju n e 11, 1970, in cash or other im m ediately available funds or in a like face am ount of T reasury bills m aturing Jun e 11, 1970. Cash and exchange tenders will receive equal treatm ent. Cash adjustm ents will be m ade for differences between the p ar value of m atur ing bills accepted in exchange and the issue price of the new bills. T he income derived from T reasury bills, w hether interest or gain from the sale or other disposition of th e bills, does not have any exemption, as such, and loss from the sale or other disposition of T reasury bills does not have any special treatm ent, as such, under the Internal R evenue Code of 1954. T he bills are subject to estate, inheritance, gift or other excise taxes, whether Federal or State, bu t are exem pt from all taxation now or hereafter imposed on the principal or interest thereof by any State, or any of the possessions of the U nited States, or by any local taxing authority. For purposes of taxation the am ount of discount a t which T reasury bills are originally sold by th e U nited States is considered to be interest. U nder Sections 454 (b ) and 1221 (5) of the Internal Revenue Code of 1954 the am ount of discount a t which bills issued hereunder are sold is not con sidered to accrue until such bills are sold, redeem ed or otherwise disposed of, and such bills are excluded from consideration as capital assets. Accordingly, th e owner of T reasury bills (other th a n life insurance com panies) issued hereunder need include in his income tax retu rn only the difference between the price paid for such bills, w hether on original issue or on subsequent purchase, and the am ount actually received either upon sale or redem ption a t m aturity during the taxable year for which the return is made, as ordinary gain or loss. T reasury D epartm ent Circular No. 418 (current revision) and this notice, prescribe the term s of the Treasury bills and govern the conditions of th eir issue. Copies of th e circular m ay be obtained from any Federal Reserve B ank or Branch. In accordance with the above announcement, tenders will be received at this bank and its branches at El Paso, Houston and San Antonio up to twelve-thirty p.m., Central Daylight SaviEig Time, Monday, June 8, 1970. Tenders may not be entered by telephone. Yours very truly, P. E. Coldwell _______________ President LAST PREVIOUS OFFERING OF TREASURY BILLS Amount, Range and Approxim ate Yield of A ccepted T enders 91-Day Bills 182-Day Bills D ue Septem ber 3, 1970 D ue D ecem ber 3, 1970 $2,743,000,000_______________________________T otal Applied F o r_______________________________ $2,808,280,000 $ 1,800,460,000________________________________ T otal A ccepted_________ ________ — i~tt ______ $1,305,620,000 P rice Y ield P rice Yield 98.294 6.749% _________________________ H igh_______________ L - - - 96.555___________ 6.814% 98.266------------------ 6.860% -------------------------------------- L o w ______________________96.530___________ 6.864% 98.275 6.824% ( 1 ) ------------------------------ Average____________________ 96.533___________ 6.858% (1) ( 1 ) These rates are on a b a n k discount basis. T h e equivalent coupon issue yields are 7 .0 4 % for th e 91-day bills, and 7 .2 0 % for th e 182-day bills. (See reverse for tender form) This publication was digitized and made available by the Federal Reserve Bank of Dallas' Historical Library (FedHistory@dal.frb.org) TENDER FOR TREASURY BILLS 182 DAYS TO MATURITY Maturing December 10,1970 Dated June 11,1970 To: Federal Reserve Rank, Station K, Dallas, Texas 75222 or — The_________________________.Branch El Paso 79999 Houston 77C01 San Antonio 78206 (D ate) Pursuant to the provisions of Treasury Department Circular No. 418 (current revision) and the provisions of t h e public announcement issued by the Treasury Department, the undersigned offers to purchase Treasury bills in the am ount shown below, and agrees to pay for the amount allotted, on or before the issue date, by the method and at the rate indicated. NONCOMPETITIVE TENDER $____________________________________ NOT TO EXCEED $200,000 Noncompetitive tenders for $200,000 or less from any one bidder, without stated price, will be accepted in full at the a v era g e price (in three decimals) of accepted competitive bids. COMPETITIVE TENDERS $ ■ .......... (ft) $ @ -" Prices should be e x pressed on the basis of 100, with not more than th ree decim als places e. g., 99.925. Fractions must not be used. * $ Bgr3 TENDERS MAY NOT BE ACCEPTED BY TELEPHONE. TENDERS BY WIRE, IF RECEIVED BEFORE T H E CLOSING HOUR, ARE ACCEPTABLE. Denominations Desired N um b er of Pieces Payment for this issue of bills cannot be made b y credit to Treasury Tax and Loan Account. METHOD OF PAYMENT M a tu rity V alue -@ $ 10,000 $_ □ 50,000 $$ 100,000 $_ $ 500,000 $_ By maturing bills held by--------------------------------------Payment to be made by------------------- -- -@ $ □ □ Charge our reserve account on payment date \| \| Draft enclosed (E ffectual delivery o f enclosed d r a f t _@ $1,000,000 $_ Delivery Instructions: □ Hold in Custody Account—Member banks for own account only □ Pledge to secure Treasury Tax and Loan Account □ Ship to------------------------------------- shall be on la te st day w hich will p e rm it p re s e n tm e n t i n o rd e r to o b ta in irrevocably collected fu n d s on p a y m e n t date) (S u b scrib er’s fu ll n am e o r co rp o ra te title ) (A ddress) By(A uthorized official s ig n a tu re a n d title ) (F o r th e acco u n t of, if te n d e r is fo r a n o th e r subscriber) (A ddress) IMPORTANT 1. No tender for less than $10,000 will be considered and each tender must be for an amount in multiples of $10,000 (m atu rity value). 2. Tenders should be forwarded in an envelope clearly addressed to this bank or appropriate branch as Fiscal Agent of t h e United States, with notation on the envelope reading “TENDER FOR TREASURY OFFERING”. Since envelopes received with this legend will not be opened until after the closing time specified in the public announcement, com m uni cations relating to other matters should not be enclosed. Envelopes for submitting tenders may be obtained from t h is bank or appropriate branch. 3. Any qualified or conditional tender will be rejected. 4. If a corporation makes the tender, the form should be signed by an officer of the corporation authorized to m a k e t h e tender and the signing of the form by an officer of the corporation will be construed as a representation by him that h e has been so authorized. If the tender is made by a partnership it should be signed by a member of the firm, w h o should sign in the form “___________________________ , a copartnership, by------------------------------------------------------------- _ a member of the firm.” 5. Tenders from those other than incorporated banks and trust companies or responsible and recognized dealers in in v e st ment securities will be disregarded, unless accompanied by a deposit of 2 percent of the total amount (maturity v a lu e) of the Treasury bills applied for, or unless the tenders are accompanied by an express guaranty of full payment by an incorporated bank or trust company. '* 6. If the language of this form is changed in any respect, which, in the opinion of the Secretary of the Treasury ’ material, the tender may be disregarded. (See reverse for announcement)	https://fraser.stlouisfed.org/title/district-notices-federal-reserve-bank-dallas-5569/new-offering-treasury-bills-542942/fulltext
Rapid versus slow rate of advancement of feedings for promoting growth and preventing necrotizing enterocolitis in parenterally fed low‐birth‐weight infants - Kennedy, KA - 1998 \| Cochrane Library Infant, Very Low Birth Weight; Enteral Nutrition [adverse effects, methods]; Enterocolitis, Necrotizing [epidemiology, etiology, prevention & control]; Incidence; Infant, Low Birth Weight [growth & development]; Infant, Premature [growth & development]; Infant, Premature, Diseases [etiology, prevention & control]; Parenteral Nutrition [adverse effects]; Randomized Controlled Trials as Topic Rapid versus slow rate of advancement of feedings for promoting growth and preventing necrotizing enterocolitis in parenterally fed low‐birth‐weight infants Information DOI: https://doi.org/10.1002/14651858.CD001241 Copy DOI Database: Cochrane Database of Systematic Reviews Version published: 26 October 1998 see what's new Type: Intervention Stage: Review Cochrane Editorial Group: Cochrane Neonatal Group Copyright: Copyright © 2008 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd. Article metrics Altmetric: Referenced in 1 policy sources Authors Kathleen A Kennedy Correspondence to:Pediatrics, University of Texas at Houston Medical School, Houston, USA kathleen.a.kennedy@uth.tmc.edu More by this author on the Cochrane Library Jon E Tyson Pediatrics, University of Texas at Houston Medical School, Houston, USA Contributions of authors Both authors participated in identifying trials for consideration and in describing the main results and conclusions. Criteria for inclusion of studies were discussed prior to searching.Each author evaluated whether the trial met eligibility criteria for inclusion in the meta‐analysis.Each author independently extracted data from the trials and evaluated their methodologic quality. Declarations of interest None Version history Published Title Stage Authors Version 2021 Aug 24 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewSam J Oddie, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub8 Revision date This is the date when the review was published Event Description2020 Oct 20New search has been performedThis updates the review 'Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants' (Oddie 2017).2020 Oct 20New citation required but conclusions have not changedThe updated search identified four new trials for inclusion (Fayyaz 2020;Hasshemi 2018;Montealegre‐Pomar 2021;Saha 2019). 2017 Aug 30 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewSam J Oddie, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub7 Revision date This is the date when the review was published Event Description2017 Apr 27New citation required but conclusions have not changedThe updated search identified 1 new trial for inclusion (SIFT 2016). New data and an increased total number of participating infants (from 949 to 3753) narrowed confidence intervals for the estimates of effect and modified implications for practice and research2017 Apr 27New search has been performedThis updates the review "Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants" (Morgan 2015) 2015 Oct 15 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewJessie Morgan, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub6 Revision date This is the date when the review was published Event Description2015 Jul 31New citation required and conclusions have changedThe updated search identified three new trials for inclusion (Raban 2014a;Raban 2014b;Modi 2015). This increased the total number of participating infants to 949 (from 618). This update provides evidence that slow advancement of enteral feed volumes increases the risk of invasive infection in very low birth weight infants.2015 Jul 31New search has been performedThis updates the review 'Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants' (Morgan 2014b). 2014 Dec 02 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewJessie Morgan, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub5 Revision date This is the date when the review was published Event Description2014 Oct 03New search has been performedThis updates the review 'Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants' (Morgan 2013).2014 Oct 03New citation required but conclusions have not changedUpdated search in September 2014 identified one new trial for inclusion (Mukhopadhyay 2014), increasing the total number of participating infants to 618 (from 588) and narrowing confidence intervals for the estimates of effect. 2013 Mar 28 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewJessie Morgan, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub4 Revision date This is the date when the review was published Event Description2012 Dec 28New citation required and conclusions have changedUpdated search in December 2012 identified one new trial for inclusion, increasing the total number of participating infants to 588 (from 496), narrowed confidence intervals for the estimates of effect.2012 Dec 28New search has been performedThis updates the review 'Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants' (Morgan 2011a). 2011 Mar 16 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewJessie Morgan, Lauren Young, William McGuirehttps://doi.org/10.1002/14651858.CD001241.pub3 Revision date This is the date when the review was published Event Description2011 Jan 11New citation required and conclusions have changedAddition of new data, and increasing the total number of participating infants to 496, narrowed the confidence intervals for the estimates of effect and modified the implications for practice and research.2010 Dec 15New search has been performedThis updates the review "Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants" published in the Cochrane Database of Systematic Reviews, Issue 2, 2008 (McGuire 2008).Search updated in December 2010. One new trial included (Krishnamurthy 2010).New co‐authors: Jessie Morgan and Lauren Young. 2008 Apr 23 Show revisionsSlow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infantsReviewWilliam McGuire, Sarah Bombellhttps://doi.org/10.1002/14651858.CD001241.pub2 Revision date This is the date when the review was published Event Description2008 Feb 13New citation required but conclusions have not changedNew authorship: Bombell S, McGuire W2008 Feb 02New search has been performedThis updates the review "Rapid versus slow rate of advancement of feedings for promoting growth and preventing necrotizing enterocolitis in parenterally fed low‐birth‐weight infants" by Kennedy and Tyson, published in the Cochrane Database of Systematic Reviews, Issue 2, 2000 (Kennedy 2000).The title has been modified to read "Slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants" and has a new authorship of Sarah Bombell and William McGuire. Changes made to the original protocol are outlined below:1. "Slow" rate of feed advancement has been defined as daily increments up to 24 ml/kg (body weight).2. The population has been restricted to very low birth weight and very preterm infants.3. Mortality, adverse neurodevelopment, growth parameters, and infection rates have been included as outcomes of interest.Search updated December 2007. One new trial has been included (Salhotra 2004). One trial previously included has now been excluded (Book 1976).The findings and implications for practice and research of the review have not changed overall.2008 Jan 11AmendedConverted to new review format. 1998 Oct 26 Rapid versus slow rate of advancement of feedings for promoting growth and preventing necrotizing enterocolitis in parenterally fed low‐birth‐weight infants Review Kathleen A Kennedy, Jon E Tyson https://doi.org/10.1002/14651858.CD001241 Revision date This is the date when the review was published Event Description2017 May 10AmendedConverted to new review format. PICOs PICOs Population Intervention Comparison Outcome The PICO model is widely used and taught in evidence-based health care as a strategy for formulating questions and search strategies and for characterizing clinical studies or meta-analyses. PICO stands for four different potential components of a clinical question: Patient, Population or Problem; Intervention; Comparison; Outcome.	https://www.cochranelibrary.com/cdsr/doi/10.1002/14651858.CD001241/information
Infection Prevention in Acute Myeloid Leukemia HCPLive is a comprehensive clinical news and information portal that provides physicians with up-to-date specialty and disease-specific resources designed to help them provide better care to patients. At HCPLive, you will find breaking news, video interviews with physician experts, in-depth conference coverage, finance and practice management updates, insight and analysis from physician contributors, and more. Infection Prevention in Acute Myeloid Leukemia Yoav Golan, MD, MS, FIDSA, attending physician and associate professor of medicine at Tufts University School of Medicine, Boston, MA, explains risk factors for infection as well as key principles of infection prevention in patients with acute myeloid leukemia (AML). To best understand how to prevent infection in patients with AML, one has to first understand what puts them at risk for infections. There are several components to that. One is exposures, such as food, water, pets, etc. Another has to do with the fact that these patients get treatment and the disease itself makes them immunocompromised, so their immune systems are not as potent in protecting them from infections. Other factors that contribute to the risk of infection have to do with very extensive exposure to the healthcare system and having a lot of procedures and lines that violate the integrity of their mucosal surfaces. The principles of preventing infections in AML patients, in fact, to a large extent, determine the outcome of those patients as they go through therapies that can weaken their immune systems. Therefore, they are very important to understand. One of the principles is to avoid unnecessarily risky exposures, and that includes food-related issues. Raw foods, for example, have to be avoided. Well water should be avoided, as well as should some exposures to animals. For example, one should not clean the litter of his cat. Another important component is preventing exposure to sick people, and we are surrounded by a lot of sick people during the winter period, but also throughout the year. Those exposures should be prevented. In addition to that, vaccination is really important, including vaccination of those people with whom they interact in their everyday life. So, if you have a relative that has AML, it’s really important for you to get a flu shot so you don’t get the flu and don’t infect someone who cannot really cope with the flu. Other components of infection prevention include infection control and better precautions. So good hand hygiene; cleaning your hands is very important for patients, and using a mask every once in a while when going into the public, particularly during the height of treatment, is extremely important. The last component that protects our patients, and on which we are going to focus a little more today, is going to be antimicrobial prophylaxis. To understand the risk of infection in AML patients it is important to understand what’s causing this risk and particularly what’s causing neutropenia, which is the main risk factor for infection in this patient population. It is important to understand what makes these patients immunocompromised so that we can monitor that and we can prevent them from getting infected while at the height of the immunocompromised state. Neutropenia is typically defined as a neutrophil or ANC count of less than 1500, with real neutropenia typically defined as less than 1000, and severe neutropenia is defined as less than 500. Now, many of the guidelines to determine who is at the highest risk of infection use a cutoff of 100, and anyone who is expected to be lower than 100 is expected to be at high risk of an infection. The neutropenia in patients with AML is really of multifactorial causes. One of the causes of neutropenia is, in fact, the disease itself. The expansion of one clone of white cells will prevent other white cells from flourishing and the diversity of those neutrophils—not necessarily their number—would be reduced. The expansion of this clone in the bone marrow will prevent the production of new neutrophils and will result in neutropenia as well. Because AML is a disease of the immune system, the treatments of AML target the immune system and add to this immunocompromised status. Most chemotherapy medications that are used in leukemia further deplete the number of neutrophils and are the main reason for neutropenia during therapy in patients with AML. Another reason for neutropenia could be some viruses that can infect those patients, such as cytomegalovirus, that have an effect on the bone marrow that is suppressing. Another reason could be some specific infections that can fill the bone marrow and prevent neutrophils from being produced.	https://www.hcplive.com/view/infection-prevention-in-acute-myeloid-leukemia
Intracellular glutathione and bronchoalveolar cells in fibrosing alveolitis: effects of N-acetylcysteine \| European Respiratory Society Intracellular glutathione and bronchoalveolar cells in fibrosing alveolitis: effects of N-acetylcysteine J. Behr, B. Degenkolb, F. Krombach, C. Vogelmeier European Respiratory Journal 2002 19: 906-911; DOI: 10.1183/09031936.02.00204902 J. Behr Article Figures & Data Info & Metrics Abstract Extracellular glutathione deficiency and exaggerated oxidative stress may contribute to the pathogenesis of fibrosing alveolitis (FA). High-dose N-acetylcysteine (NAC) supplementation partially reverses extracellular glutathione depletion and oxidative damage, but effects on intracellular glutathione are unknown. Intracellular total glutathione (GSH t) and activation of bronchoalveolar lavage cells (BAC) obtained from 18 FA patients (9 males, aged 52±2 yrs), before and after 12 weeks of oral NAC (600 mg t.i.d.), were assessed. Eight healthy nonsmokers (2 males, aged 36±6 yrs) served as a control group. Intracellular GSH t was decreased in FA (1.57±0.20 nmol·1×10 6 BAC −1 versus 2.78±0.43 nmol·10 6 BAC −1). After NAC treatment, the intracellular GSH t content increased (1.57±0.20 versus 1.87±0.19 nmol·1×10 6 BAC −1). The spontaneous oxidative activity of BAC decreased after NAC treatment (2.7±0.8 versus 1.0±0.2 nmol·1×10 6 BAC −1·h −1). Interleukin-8 concentration (82.1±31.5 versus 80.0±22.6 pg·mL bronchoalveolar fluid (BALF), nonsignificant (ns)) and myeloperoxidase activity (1.93±0.64 versus 1.55±0.47 mU·mL −1 BALF, ns) did not change significantly, but were found to be inversely correlated to intracellular GSH t. In conclusion, high-dose N-acetylcysteine supplementation increases intracellular glutathione levels slightly. This increase is associated with a mild reduction of oxidative activity but not with a reduction of bronchoalveolar cell activation in these patients. fibrosing alveolitis glutathione interleukin-8 myeloperoxidase oxidative activity J. Behr was supported by Wilhelm Sander-Stiftung During the last 10 yrs considerable evidence has accumulated linking increased oxidative stress and reduced antioxidative capacity to the pathogenesis of fibrosing alveolits (FA) 1–8. Activated inflammatory cells enter the lower respiratory tract and release increased amounts of reactive oxygen species (ROS) 1, 2, 5, 7, leading to oxidative injury of the lung parenchyma 1, 3, 7, 8, 9. Glutathione in its reduced form (GSH) prohibits oxidant injury in healthy lungs but has been shown to be lacking in pulmonary fibrosis, at least in the extracellular compartment 4, 7, 9–11, thus aggravating the pulmonary redox imbalance. In several studies it has been demonstrated that N-acetylcysteine (NAC) administered orally or intravenously is capable of stimulating glutathione synthesis by providing cysteine residues for the γ-glutamyl cycle located in the cytoplasm, thereby partially restoring glutathione levels in the epithelial lining fluid of fibrotic lungs 9–11. This was accompanied by a decrease in oxidized methionine residues in extracellular proteins and by functional improvements in the patients in one study 9. However, glutathione is also an important intracellular antioxidant, as, by influencing the intracellular redox balance, it is linked to the activation of transcription factors, such as nuclear factor (NF)-κB or activation protein (AP)-1, and may play a crucial regulatory role in inflammatory and reparative processes relevant in lung fibrosis 12–14. Therefore, intracellular glutathione levels and parameters of bronchoalveolar inflammatory cell activation (oxidative activtivity, interleukin (IL)-8 concentrations, and myeloperoxidase (MPO) activity) in patients with FA before and after a 12-week course of high-dose NAC treatment were studied. Materials and methods Patients and protocol The patients studied and the study protocol used were identical to those described in detail in a previous study 9. In brief, a total of 20 patients (10 males, aged 52±2 yrs) were enrolled for a prospective study of NAC 600 mg t.i.d. Two patients dropped out, one because of diarrhoea and the other because of a surgical procedure not related to the study drug. Of the remaining 18 patients (9 males) who completed the study, nine suffered from idiopathic pulmonary fibrosis (histologically confirmed) and nine had collagen vascular disease with lung involvement. Thirteen of 18 patients were under maintenance immunosuppression with prednisolone (mean daily dose 6.7±1.8 mg), three of which received additional azathioprine (n=2) or cycloposphamide (n=1). The remaining five patients were off any immunosuppressive treatment because of lack of effect. All patients were nonsmokers. The study protocol included a bronchoalveolar lavage (BAL) study before and after 12 weeks of NAC therapy. Written informed consent was obtained from all patients and the protocol was approved by the local ethical committee. Controls A standard BAL was performed in eight healthy volunteers (2 males, age 36±6 yrs) without a history of atopy or bronchopulmonary disease and with normal pulmonary function tests. All controls had never smoked. BAL cell differential was within the laboratory normal range. Written informed consent was obtained from all controls and the protocol was approved by the local ethical committee. Biological samples BAL fluid (BALF) was obtained by standard techniques. Aliquots of freshly obtained bronchoalveolar cells (BAC) were taken for glutathione assays (see later), total cell counts (Coulter counter), and cytocentrifuge preparations for differential cell counts. The cells were pelleted and the supernatants were used to assess the MPO activity and IL-8 concentration. Intracellular glutathione levels Total glutathione (GSH t=GSH+2×oxidized glutathione (GSSG)) concentrations were measured using standard techniques, as reported previously 7. Immediately after the BAL procedure, cells were gently centrifuged (300× g for 10 min), the supernatant was then removed and the cells were resuspended in phosphate-buffered saline. An aliquot of 1×10 6 BAC was lysed by incubation in 1 mL of distilled water for 5 min. After centrifugation (3,000× g for 5 min), 100 µL of the lysate was mixed with 1.1 mL of 0.1 M sodium phosphate buffer, pH 7.0, containing 1 mM ethylenediamine tetraacetic acid, 0.2 mM nicotinamide adenine dinucleotide phosphate, 63.5 µM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Serva, Heidelberg, Germany), and 4 U·mL −1 glutathione reductase. The rate of reduction of DTNB was recorded spectrophotometrically at a wavelength of 412 nm. All measurements were made in triplicate and the average value was calculated. The GSH t concentration of the BAC lysate sample was calculated using an internal standard of 0.84 µM GSH 7. The GSH t content of BAC was expressed as nmol·1×10 6 BAC −1. Oxidative activity For measurement of the oxidative activity, ex vivo cultures of BAC were used as previously described 7. BAC obtained by BAL were cultured in standard medium (Roswell Park Memorial Institute (RPMI) 1640, Sigma Chemical Co., St Louis, MO, USA) using 6-well, flat-bottomed, plastic-culture dishes (Flacon, NJ, USA). After incubation for 30 min at 37°C with 5% carbon dioxide, nonadherent cells were removed, generating BAC monolayer cultures consisting mainly of alveolar macrophages (>95%) 7. For measurement of oxidative activity, BAC cultures were incubated with Hank's balanced salt solution (Sigma) containing 50 µM of GSH ±160 nM phrobol myristate acetate (PMA). Blanks without BAC were processed in exactly the same way. After 1 h of incubation the supernatant was removed and catalase (Sigma) was added to a final concentration of 80 U·mL −1 to stop hydrogen peroxide (H 2O 2)-dependent reactions 15. The samples were mixed with DTNB (final concentration 250 µM) and after 5 min of incubation the absorption was measured at 412 nm (Lambda 19 Spektrophotometer; Perkin Elmer, Ueberlingen, Germany). The concentration (nmol) of GSH, which was oxidized by 1×10 6 BAC −1·h −1 spontaneously or with PMA stimulation, was calculated from the decrease of the GSH concentration in the presence of BAC (±PMA) as compared to the blanks. All measurements were performed in duplicate. The results were expressed as nmol·1×10 6 BAC −1·h −1. Interleukin-8 The IL-8 concentration was determined in BALF supernatants using a commercially available enzyme-linked immunosorbent assay (ELISA; Endogen Human Interleukin-8 ELISA; Endogen, MA, USA). All determinations were performed in duplicate. The results are expressed as pg·mL·BALF −1. Myeloperoxidase activity MPO activity was measured as described by Suzuki et al. 15. In brief, 400 µL of fresh BALF supernatant (3,000× g for 10 min) were mixed with 420 µL of 150 mM sodium hydrogen phosphate buffer and 80 µL of 20 mM tetramethyl benzidine (Serva). After 5 min of incubation at room temperature, the reaction was started by adding 100 µL of 3 mM H 2O 2 and absorption was recorded by spectrometer at 655 nm. All measurements were performed in duplicate. The results are expressed as mU·mL −1 BALF. Statistics Data are expressed as mean±sem. For statistical analysis, t-tests for dependent or independent samples were employed, as appropriate. Correlations were calculated according to Pearson correlation coefficient. p-Values <0.05 were considered significant. Results Bronchoalveolar lavage differential cell counts The results of absolute and relative cell counts are summarized in table 1⇓. Total cell count and absolute numbers of alveolar macrophages, neutrophils, eosinophils and lymphocytes were significantly elevated in FA patients, both before and after NAC treatment, compared with healthy controls. The percentage of alveolar macrophages decreased in FA patients before and after NAC, as compared to the control group, whereas the percentages of neutrophils, eosinophils and lymphocytes increased. In contrast, absolute and relative cell counts did not differ significantly between FA patients before and after NAC treatment (table 1⇓). Table 1— Differential cell counts in bronchoalveolar lavage fluid (BALF) in controls and in subjects with fibrosing alveolitis (FA) before and after N-acetylcysteine (NAC) treatment Glutathione content The GSH t content of BAC from patients with FA before NAC treatment was significantly lower compared to healthy nonsmokers (1.57±0.20 nmol·1×10 6 BAC −1 versus 2.78±0.43 nmol·1×10 6 BAC −1, p<0.05). This finding of a 44% lower cellular GSH t content in FA patients may, at least in part, be due to the different composition of total BAC from controls and FA patients, particularly the 23.4% lower percentage of alveolar macrophages in FA patients before NAC therapy. It should be noted that the control group was younger than the patient group. However, there was no relationship between age and intracellular GSH t in the control and patient groups, and no reports were found in the literature. After 12 weeks of NAC supplementation, there was a significant increase of the GSH t content of BAC from FA patients (1.57±0.20 nmol·1×10 6 BAC −1 versus 1.87±0.19 nmol·1×10 6 BAC −1, p<0.05). Although intracellular GSH t did not normalize, the difference was no longer significant when compared with controls (p=0.082). As shown in figure 1, 13 out of 18 patients (72%) had higher intracellular GSH t levels after NAC treatment, one (6%) remained unchanged, and four (22%) experienced a small decline of intracellular GSH t (fig. 1⇓). This finding cannot be explained by different BAC compositions, because there was no significant change of absolute or relative BAC numbers during the treatment period (table 1⇑). <here is a image d1d6993dfde23ab3-14397a148d340428> Fig. 1.— The total glutathione (GSH t) contents of bronchoalveolar cells (BAC) of 18 patients with FA before and after N-acetylcysteine (NAC). Data are presented as mean±sem. : p<0.05. The extracellular glutathione concentrations in these patients have been previously published 11. The concentrations of GSH t and GSH increased significantly during the 12-week course of NAC supplementation 11. Interestingly, there was a significant positive correlation between GSH t concentration in the epithelial lining fluid and the glutathione content of BAC (r=0.46; p<0.005). Oxidative activity of bronchoalveolar cells The spontaneous oxidative activity of ex vivo cultivated BAC declined significantly from 2.70±0.83 nmol·1×10 6 BAC −1·h −1 to 1.02±0.21 nmol·1×10 6 BAC −1·h −1 (p<0.05) after NAC treatment, whereas the PMA-stimulated oxidative activity was only slightly diminished (8.26±1.32 nmol·1×10 6 BAC −1·h −1 versus 7.13±1.00 nmol·1×10 6 BAC −1·h −1, nonsignificant (ns)) (fig. 2⇓). As BAC composition did not change significantly during NAC treatment (table 1⇑) 11, these changes do not appear to be attributable to differences in BAC composition, athough in this study it was not possible to establish which cell population(s) was (were) responsible for the reduction in oxidative activity. Correlation analysis showed a slight trend toward an inverse relationship between intracellular GSH t and spontaneous or PMA-stimulated oxidative activity of BAC (r=−0.277, ns, and r=−0.237, ns, respectively). <here is a image c0d953029191739f-c8252bf0e016e791> Fig. 2.— Spontaneous (└) and phorbol myristate acetate-stimulated (□) oxidative activity of ex vivo cultivated bronchoalveolar cells (BAC) before and after N-acetylcysteine (NAC) treatment. For further details see text. : p<0.05 pre- versus post-NAC treatment. Interleukin-8 concentrations As reported previously 16, 17, IL-8 concentrations in BALF were significantly increased in FA patients when compared to healthy controls (82.1±31.5 pg·mL·BALF −1 versus 18.1±2.0 pg mL·BALF −1, p<0.01). The IL-8 concentrations in native BALF were not significantly altered after NAC treatment (82.1±31.5 pg·mL·BALF −1 versus 80.0±22.6 mL −1 BALF, ns). However, a significant inverse correlation existed between the GSH t content of BAC and IL-8 levels in BALF, as shown in figure 3⇓ (n=36, r=0.45, p<0.05). <here is a image ccae3b6518b27e05-3c7501f1fa2b5b21> Fig. 3.— Inverse correlation between intracellular total glutathione (GSH t) content of bronchoalveolar cells (BAC) and interleukin (IL)-8 concentration in bronchoalveolar lavage fluid (BALF) in 18 patients with FA before and after N-acetylcysteine treatment. Myeloperoxidase activity MPO is a marker enzyme of neutrophilic inflammation and is responsible for enhanced oxidative stress mediated by hypohalides. MPO activity in native BALF was slightly reduced after NAC supplementation (1.93±0.64 mU·mL·BALF −1 versus 1.55±0.47 mU·mL·BALF −1, ns). However, a significant inverse correlation was observed between GSH t content of BAC and MPO activity in native BALF (n=36, r=0.54, p<0.01) (fig. 4⇓). <here is a image d712766126ed9d14-e452f1f2c8f4f8ff> Fig. 4.— Inverse correlation between intracellular total glutathione (GSH t) content of bronchoalveolar cells (BAC) and myeloperoxidase (MPO) activity in bronchoalveolar lavage fluid (BALF) in 18 patients with FA before and after N-acetylcysteine treatment. Discussion The most important finding in this study was that glutathione deficiency in FA was not restricted to the extracellular compartment as reported previously 4, 7, 9–11, but that there was also an intracellular glutathione deficiency in BAC. However, it was not possible to establish whether the lack of intracellular glutathione in BAC cells was restricted to a specific cell population or was caused by the presence of apoptotic cells. It was also unclear whether the lower GSH t content of BAC was the cause or consequence of BAC activation in FA. Despite the limitations of this study, the finding that intracellular GSH t content in BAC was reduced in FA patients is of interest, as intracellular glutathione is linked to important immunological, inflammatory, and reparative cell functions and may have relevant consequences for the course of the disease 12–14, 18, 19. Therefore, it is of particular interest that oral NAC supplementation, at a high dose of 1,800 mg·day −1 over a period of 12 weeks, was able to significantly increase intracellular GSH t, although it did not normalize GSH t levels. These findings parallel previous reports, which showed that NAC increases extracellular glutathione levels in patients with pulmonary fibrosis 9–11. A significant positive correlation between intra- and extracellular glutathione levels was observed. This data, therefore, provides additional confirmation that NAC works as a glutathione precursor not only in the liver but also in the lungs. Previous reports on the anti-inflammatory actions of NAC suggest it has an antioxidative effect and may play a role as a glutathione precursor 14, 18, 20–22. However, there are conflicting reports regarding the effect of oral NAC on oxidant release from phagocytes 20, 23. In this study, there was a significant drop in the spontaneous oxidative activity of BAC after 12 weeks of NAC treatment, whereas PMA-stimulated oxidative activity did not change significantly. This could be due to slight anti-inflammatory actions of NAC. The findings in this study parallel the results of Borok et al. 24, who also observed a decrease in spontaneous superoxide anion release by alveolar macrophages after 3 days of glutathione aerosol administration (600 mg every12 h) in idiopathic pulmonary fibrosis patients. The data from this study suggests that the decrease of spontaneous oxidative activity is linked, at least indirectly, to the increase of intracellular GSH t after NAC supplementation, although it is not clear whether it is the cause or the consequence. It has been well documented that oxidative stress plays a key role in the regulation of IL-8 secretion 25. The finding that IL-8 concentrations in BALF are inversely correlated to intracellular GSH t levels supports the idea that increased oxidative stress triggers IL-8 gene expression and secretion. However, NAC induced an average increase of intracellular GSH t of 19%, which was insufficient to induce a significant reduction in IL-8 concentrations in BALF. MPO is a marker of neutrophil activation and was used here as an indicator of neutrophil inflammatory activity, which could be modified by altered intra- and/or extracellular glutathione levels. A decrease in the MPO concentration in serum from smokers after 8 weeks of treatment with NAC (200 mg t.i.d.) has been reported previously 20. The reduction in MPO activity in BALF observed in this study was statistically ns. Nonetheless, this data suggests that MPO release is inversely linked to intracellular glutathione. Although evidence of a causal relationship between intracellual GSH t and IL-8 or MPO secretion could not be generated, the present study's results are in agreement with this notion. To conclude, the data in this study has shown for the first time, that intracellular total gluthione is decreased in fibrosing alveolitis and that this decrease is partially reversed by high-dose oral N-acetylcysteine supplementation. Despite the limitations of the study, the observations are consistent with the hypothesis that an increase in intracellular glutathione, by virtue of its antioxidative effect, may have a slight anti-inflammatory effect on bronchoalveolar cell activation, as measured by spontaneuos oxidant release. The known pathways of oxidative activation of transcription factors and key cytokines, such as interleukin-8, provide the pathophysiological background for profound anti-inflammatory actions of anti-oxidative therapy 13, 14, 18, 19, 25. However, significant changes in interleukin-8 and myeloperoxidase levels after N-acetylcysteine supplementation were not shown, although intracellular total glutathione content was inversely correlated with these pro-inflammatory mediators. Overall, the changes of inflammatory cell activation observed were small and may be clinically nonsignificant. However, prolonged treatment or the use drugs that are more effective in elevating intracellular glutathione, may provide new and more potent anti-inflammtory treatment strategies. Acknowledgments The authors would like to thank B. Braun and A. Neuber for expert technical assistance. Received January 15, 2001. Accepted November 2, 2001. © ERS Journals Ltd References ↵ Cantin AM, North SL, Fells GA, Hubbard RC, Crystal RG. Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis. J Clin Invest 1989; 79: 1665–1673. ↵ Clement AK, Chadelat K, Masliah J, et al. A controlled study of oxygen metabolite release by alveolar macrophages from children with interstitial lung disease. Am Rev Respir Dis 1987; 136: 1424–1428. ↵ Behr J, Maier K, Krombach F, Adelmann-Grill BC. Pathogenetic significance of reactive oxygen species in diffuse fibrosing alveolitis. Am Rev Respir Dis 1991; 144: 146–150. ↵ Cantin AM, Hubbard RC, Crystal RG. Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am Rev Respir Dis 1989; 139: 370–372. ↵ Strausz J, Müller-Quernheim J, Steppling H, Ferlinz R. Oxygen radical production by alveolar inflammatory cells in idiopathic pulmonary fibrosis. Am Rev Respir Dis 1990; 141: 124–128. Meyer A, Buhl R, Kampf S, Magnussen H. Intravenous N-acetylcysteine and lung glutathione of patients with pulmonary fibrosis and normals. Am J Respir Crit Care Med 1995; 152: 1055–1060. Dröge W, Schultz-Osthoff K, Mihm S, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J 1994; 8: 1131–1138. Suzuki K, Ota K, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 1983; 132: 345–352. Winyard PG, Blake DR. Antioxidants, redox-regulated transcription factors, and inflammation. Adv Pharmacol 1997; 38: 403–421. Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999; 21: L1067–L1088. Sala R, Moriggi E, Corvasce G, Morelli D. Protection by N-acetylcysteine against pulmonary endothelial cell damage induced by oxidant injury. Eur Respir J 1993; 6: 440–446. Mulier B, Rahman I, Watchorn T, MacNee W, Jeffery PK. Hydrogen peroxide-induced epithelial injury: the protective role of intracellular nonprotein thiols (NPSH). Eur Respir J 1998; 11: 384–391. Drost E, Lannan S, Bridgeman MME, et al. Lack of effect of N-acetylcysteine on the release of oxygen radicals from neutrophils and alveolar macrophages. Eur Respir J 1991; 4: 723–729. Borok Z, Buhl R, Grimes GJ, et al. Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. Lancet 1991; 338: 215–216. Intracellular glutathione and bronchoalveolar cells in fibrosing alveolitis: effects of N-acetylcysteine J. Behr, B. Degenkolb, F. Krombach, C. Vogelmeier	https://erj.ersjournals.com/content/19/5/906.long
photos every 5 min during the day to aid in devel \| Download Scientific Diagram Download scientific diagram \| photos every 5 min during the day to aid in devel from publication: mHealth Applications Use and Potential for Older Adults, Overview of \| mHealth and Telemedicine \| ResearchGate, the professional network for scientists. photos every 5 min during the day to aid in devel Full-text available Jan 2020 Zaki Ansaar Jamil Hussain Jae Hun Bang [...] Kim Young Woo Citations ... All stakeholders in our study recommended to create the ActiveHipþ mobile application following a user-friendly mobile app design, with useful content for communication and information sharing, which has been reported in previous work analysing the implementation of mHealth systems in different health conditions [26,39]. The need to develop a user interface design with appropriate visual objects, such as large buttons that can be distinguished from other resources, or the use of font size adjusted to the needs of older adults were previously highlighted [40] . The participants in our study also made suggestions for the app to increase uptake of health technology and reduce the frustration during the use of the app [26,41], which was previously tied to the need to develop a clear navigational structure and an interface aligned with the expectations of older adults [40,42]. ... ... The need to develop a user interface design with appropriate visual objects, such as large buttons that can be distinguished from other resources, or the use of font size adjusted to the needs of older adults were previously highlighted [40]. The participants in our study also made suggestions for the app to increase uptake of health technology and reduce the frustration during the use of the app [26,41], which was previously tied to the need to develop a clear navigational structure and an interface aligned with the expectations of older adults [40, 42]. The specific recommendations for older adults with hip fracture were mainly focussed on the contents and the delivery modes such as, short videos, infographics, positive messaging and older adults stories to improve user experiences [43]. ... Co-creation of mHealth intervention for older adults with hip fracture and family caregivers: a qualitative study Article Oct 2022 Patrocinio Ariza-Vega Rafael Prieto-Moreno Marta Mora-Traverso Pablo Molina-García Miguel Martín-Matillas Purpose: Hip fracture results in an older person's loss of independence. Limited healthcare resources make mobile Health (mHealth) an alternative. Engaging key stakeholders in health technology development is essential to overcome existing barriers. The aim of this study was to establish perspectives of older adults with hip fracture, family caregivers and health professionals (stakeholders) on the development of a mHealth system. Materials and methods: Qualitative study guided by user-centered design principles with focus groups to engage stakeholders during the development. Seven focus groups were conducted [older adults with hip fracture (n = 2), caregivers (n = 3), and health providers (n = 2)] with 45 participants (14 older adults, 21 caregivers and 10 health providers). Inclusion criteria were older adults ≥ 65 years who sustained a hip fracture in the previous 3 months; family caregiver of a person with hip fracture; and health providers with 2+ years of clinical experience working older adults with hip fracture. We followed standard methods for focus groups, including recording sessions, transcription and conducting an inductive content analysis. The same moderator, with clinical and research experience, conducted all focus groups. Results: Three themes were generated to consider for a future mHealth intervention: (1) user-friendly design; (2) content to include recovery and prevention information; and (3) implementation factors. Our mHealth system was developed based on feedback from participants. Conclusions: Co-creating mHealth technology with stakeholders is essential for uptake and adherence. We provide an overview of the development of ActiveHip+, an mHealth system for the clinical care of older adults with hip fracture. ... The specificities inherent to mHealth applications for the elderly, emphasize the need to know well the profile of older adult to be achieved with the product to be developed. Thus, we must think about the forms to involve this profile in the designing of these applications [9], not only to deal with graphic elements (i.e., font size, button colors and position of the elements) [10] but also to project the interaction flow [11] in order to optimize the cognitive process of the target audience. ... Mobile applications for elderly healthcare: A systematic mapping Article Full-text available Jul 2020 PLOS ONE Joseane O. V. Paiva Rossana M. C. Andrade Pedro Almir M. de Oliveira Paulo Duarte Ivana Cristina de H. C. Barreto Context The increase in the population aging has brought more significant concern about how proper care will be provided to the elderly in the future. Thus, the development of technological solutions for the health domain has gained more prominence. Joining this scenario to the growing use of mobile devices for daily activities, several mobile applications focused on the elderly healthcare have been developed with healthcare and software engineer professionals involved. However, there is no survey to help both professionals to take decisions on the target of application, elderly profile, empirical validation techniques, among others. Thus, the following question arises: how have mobile applications for elderly healthcare been addressed in the literature in the past years? Objective To identify the state of the art in the literature concerned with the development of mobile applications for elderly healthcare, considering healthcare and software Engineering viewpoints. Method We performed a systematic mapping conducted by health and software engineering researchers to provide an interdisciplinary investigation of the papers that address mobile applications for elderly healthcare, summarizing the data collected under the following classification: target of application, older adult profile, spatial-temporal distribution, techniques for empirical validation and type of software engineering research. Results We found a total of 2533 papers and, after applying our eligibility criteria, we got 149. We observed aspects related to the digital health initiative type, using the classification proposed by the World Health Organization (WHO), the elderly profile prioritized by the application, the spatial-temporal distribution of the studies, the empirical validation type, and the research contribution of each analyzed paper to the software engineering area. Conclusions Regarding the WHO classification, we noticed that two categories were more frequently found, Clients and Data Services, and that none of the mobile apps were classified in the Health System Manager category. The data extraction result also reveals that most of the applications found in the literature focused on the independent elderly. Moreover, we observed that most of the studies were proposals of solutions for elderly health and the validation process of these solutions generally consisted of controlled experiments and usability evaluations. At last, the research focused on mobile applications for elderly healthcare has been performed mostly by developed countries. ... Additionally, approximately 46% of clinicians reported planning to use mHealth apps in their practice [24], believing apps can improve their knowledge and interactions with patients. The development and use of mHealth apps supporting selfmanagement of more chronic health problems and for improving communication between clinicians and patients is recommended as an opportunity for increasing changes in patient behavior and better health outcomes [22, 25] . Although mHealth apps are available for managing urinary incontinence [13,14,26], there currently are none for ABL. ... Self-management of accidental bowel leakage and interest in a supportive m-Health app among women Article Dec 2019 INT UROGYNECOL J Donna Z. Bliss Olga V. Gurvich Sunita Patel Isuzu Meyer Holly E. Richter Introduction: Self-management with clinician guidance is a cornerstone of successful conservative treatment for accidental bowel leakage (ABL). There are currently few resources to assist patients with that essential self-management. The purpose of this study was to describe self-management of ABL and explore interest in a mobile health application (m-Health app) for supporting ABL self-management among community-living women. Methods: Using an observational/descriptive design, women (18+ years) previously seen in a urogynecology clinic for ABL were mailed a survey containing eight multiple-choice questions and an open-ended prompt for comments. Results: Survey responses were received from 161 women (18% response rate). The highest percentage of participants was aged 61-70 years (39%). Nearly half of the participants (47%) "did not know anything" about ABL self-management before visiting a clinician. Only 4% "knew a lot." Of those who have been trying to self-manage their ABL (n = 132), 37% reported that it was not effective, and only 5% thought their self-management was "very effective." Half (50%) of the participants had "a lot of" interest in an m-Health app to support managing ABL, and 30% had "some" interest. The vast majority (89%) thought that it was "very important" to have ongoing guidance and support for self-managing ABL. Conclusions: Before visiting a clinician, most women with ABL lacked knowledge about ABL self-management, and their self-management was not very effective. Women thought having guidance to self-manage ABL was important. There would be good interest among women in an m-Health app supporting self-management of ABL if one were created. ... Some communities are creating resource directories so both health professionals and older adults will know where they can safely exercise and/or join physical activity programs. 39 Additionally, there is often a lack of coordination between community and clinical settings resulting in an imbalance in supply and demand for community based interventions. A recommended solution is having dedicated programming so that evidence-based interventions are consistently offered and routinely updated in the community and health professionals will feel comfortable routinely referring their patients to such interventions. ... Best Practices for Ensuring a Healthy and Active Lifestyle in Older Adults Chapter Jan 2019 Marcia G Ory Barbara Resnick Mark Stoutenberg Aya Yoshikawa Matthew Lee Smith ... Although various mHealth apps exist that target chronic illnesses and diseases prevalent among the older adult population, many of these apps are not considered usable by older users [8] . Various studies report challenges experienced during interactions with interfacebased systems, including those expressed by the older adult population [8,9,10,11]. ... ... Although various mHealth apps exist that target chronic illnesses and diseases prevalent among the older adult population, many of these apps are not considered usable by older users [8]. Various studies report challenges experienced during interactions with interfacebased systems, including those expressed by the older adult population [8, 9,10,11]. Existing mobile apps are often perceived to be too complex [9] [11], move at a pace not conducive to users who require a longer learning time [10], or were not legible to the decreased visual acuity of many older users [9]. ... ... As a result of varying usability challenges, many older adults report abandoning and never adopting potentially useful mHealth apps. Use of mHealth apps decreases with age among adults 60 and older, with only 10% of adults aged 65 and older choosing to adopt the use of these apps [8] . Despite such low use, 42% of older adults reported an interest in utilizing such as technology in a national survey addressing the current use and interest in using mHealth apps [12]. ... Universally Accessible mHealth Apps for Older Adults: Towards Increasing Adoption and Sustained Engagement Conference Paper May 2017 Christina N. Harrington Ljilja Ruzic Jon A. Sanford Mobile health (mHealth) applications are emerging as a convenient approach to monitoring and tracking health informatics and promoting health-related behaviors. Despite proven health benefits of these systems, many are not designed in consideration of older users. Studies report low or inconsistent usage and high abandonment among these applications, often attributed to poor motivation and engagement strategies utilized in the workflow of the application. However, we posit that a lack in the universal access of a mHealth interface may also contribute to low and inconsistent usage, and is an equally important factor of consideration when designing a product. The approach of universal design should be applied when designing, developing, and evaluating mHealth applications as a consideration to making products usable by the general population. The universal design mobile interface guidelines support the design and development of mobile applications that are accessible by all users equally. As an adaptation to these guidelines, we outline a tool for evaluating mHealth applications for usability and perceived usefulness. We also discuss the importance of universally designed interfaces in the adoption and sustained engagement of mHealth applications. Future work in this research area aims to utilize this approach in the evaluation of mHealth apps that target physical activity. This paper contributes to the field of human-computer interaction as it better positions mHealth applications to be adopted by a wider subset of the general population, increasing the likelihood of individuals experiencing associated health-related benefits. Note: Assessing Cancer Patient Usability of a Mobile Distress Screening App Conference Paper Jun 2022 Alyssa Donawa Christian Powell Rong Wang Ming-Yuan Chih Corey E. Baker View	https://www.researchgate.net/figure/photos-every-5-min-during-the-day-to-aid-in-devel_fig1_303098318
Monitoring the evolution of material structure in cement pastes and concretes using electrical property measurements \| Request PDF Request PDF \| On Dec 1, 2013, K. B. Sanish and others published Monitoring the evolution of material structure in cement pastes and concretes using electrical property measurements \| Find, read and cite all the research you need on ResearchGate Monitoring the evolution of material structure in cement pastes and concretes using electrical property measurements December 2013 Construction and Building Materials49(3):288-297 DOI: 10.1016/j.conbuildmat.2013.08.038 Authors: <here is a image f2f62089ad6ff61c-d19572ff1996375a> K. B. Sanish K. B. Sanish <here is a image 4f4c4746e3e74a31-327c945ba8eab1d4> Narayanan Neithalath Arizona State University <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam Indian Institute of Technology Madras ... The initial zeta potential of CSH phases is negative, but may become positive as the Ca 2+ concentration in the cement pore solution increases and Ca 2+ adsorb onto the surface of CSH phases [33,34]. Ettringite and monosulfate show positive zeta potentials, and syngenite, portlandite and gypsum show zero or negative zeta potentials [35] . Thus, the measured zeta potential of cement suspension is an average value of all the particles presented in the suspension. ... ... Thus, the measured zeta potential of cement suspension is an average value of all the particles presented in the suspension. Conductivity of cement paste depends on the ionic concentration and ionic conductivity [35] . Under the effect of cement hydration, conductivity and zeta potential of cement paste increased with time. ... ... It can be seen that the growth of s d was two orders of magnitude lower than that of s s , and growth of s d gave different orders of thixotropic a w/c=0. 35 (b)w/c=0.45 behavior for different pastes. ... Effect of mineral admixtures on the structural build-up of cement paste Article Jan 2018 CONSTR BUILD MATER <here is a image da715728fb66359d-80f1eb03dcdf836f> Qiang Yuan <here is a image 61da1223e96b4402-eab647a852369328> Dajun Zhou Baiyun Li <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Caijun Shi Structural build-up is of great importance in many concrete applications, such as formwork pressure, multi-layers casting, slip form paving, stability, and 3D printing. In this paper, the growth of static yield stress (τs) and thixotropic area were used to characterize the structural build-up of cement paste. The effects of different mineral admixtures, i.e. silica fume (SF), ground slag (GS), fly ash (FA), attapulgite (AG), nano calcium carbonate (NC), and nano silica (NS) on the structural build-up of cement pastes were studied. Calorimetric curves, zeta potential and conductivity of the pastes were also measured. Results showed that thixotropic behavior characterized by the growth of τs and thixotropic area gave different ranking on cement pastes with various mineral admixtures. NS, AG, NC and SF increased the growth of τs over 120 min, and NS was the most effective one. FA and GS decreased the growth of τs over 120 min. Hydration of cement played a dominant role in the structural build-up of cement paste. However, high hydration rate didn't always lead to high structural build-up rate. ... Electrical resistivity of concrete is tool that can be used in various scenarios, including rapid chloride intrusion, setting time, early age characteristics, fiber orientation and compressive strength amongst others (Farooq 2009 , Sanish 2013 , Azarsa 2017. As previously mentioned, electrical resistivity is a product of the pore solution within concrete. ... ... Many studies have investigated the development of electrical resistivity and conductivity as concrete and cementitious materials age (Hansson 1983, Li 2007, Sant 2010 , Sanish 2013 , Sharisha 2017). These studies found a steep increase in resistivity initially, which then plateaus similar to the compressive strength of concrete as age of concrete increases. ... Non-destructive Evaluation of Concrete using Electrical Resistivity and Ultrasonic Wave Propagation Article May 2022 Justin Harris With the intent to investigate the relationship between concrete physical properties and non-destructive evaluation techniques (NDE), several experiments were performed. Specimens were made at varying geometries using a range of different concrete mixes under several different curing conditions. These specimens were subjected to a combination of electrical resistivity and ultrasonic wave propagation measurements. One part of this study investigated determining the orientation of steel fibers using electrical resistivity. This resulted in the fabrication of a four-probe square device with the potential capabilities of determining fiber orientation. The other part of this research applied ultrasonic wave propagation via through-transmission along with electrical resistivity via the uniaxial method. The results from this uncovered an exponential relationship between the pulse wave velocity and formation factor for saturated specimens. With the formation factor’s relationship to strength and microstructural properties, this relationship may lead to predicting strength and pore structure using relationships to a simple ultrasonic property. ... Since the conductivity of solid and vapor phases are several orders of magnitude lower than the liquid phase [48] [49][50], it is assumed that the electrical conduction in the cement matrix only occurs through its liquid phases. Therefore, ion concentration in the liquid phase and tortuosity of cement microstructure are two key factors affecting the electrical resistivity of the cement system, which can be expressed by Equation (2) [51]. ... ... This result indicates that resistivity extracted from the EIS test can be regarded as a real-time monitoring parameter and applied to measure the hydration heat of mass concrete structures in the future. Since the conductivity of solid and vapor phases are several orders of magnitude lower than the liquid phase [48] [49][50], it is assumed that the electrical conduction in the cement matrix only occurs through its liquid phases. Therefore, ion concentration in the liquid phase and tortuosity of cement microstructure are two key factors affecting the electrical resistivity of the cement system, which can be expressed by Equation (2) [51]. ... Nondestructive Monitoring Hydration of Belite Calcium Sulfoaluminate Cement by EIS Measurement Article Full-text available Jun 2022 Mian Wang Zhuolin Wang Junjie Li <here is a image 9eb7efc4d08d5d61-09f2e0dd49fd0571> Lin Chi In this study, the impact of water-to-cement (w/c) ratios of belite calcium sulfoaluminate cement (BCSA) on the hydration kinetics and the electrochemical impedance spectroscopy (EIS) parameters is studied. According to the analysis of classic hydration measurements, such as calorimetry tests, chemical shrinkage content, and chemically bound water content, it can be concluded that a higher w/c ratio clearly accelerates the hydration of BCSA cement paste. The electrical resistivity of BCSA0.35 cement paste is more than 4.5 times that of BCSA0.45 and BCSA0.5, due to the gradually densified micropore structure blocking the electrical signal transmission rather than the free charged-ion content. The porosity of BCSA0.5 is 27.5% higher than that of BCSA0.35 and 7.8% higher than that of BCSA0.45, which proves the resistivity is clearly related to the variation in microstructure, especially for the porosity and pore size distribution. The novelty of this study is the linear regression with logarithm terms of electrical resistivity and classic hydration parameters such as chemical shrinkage, cumulative hydration heat, and chemically bound water is established to extend the classical expression of cement hydration degree. It indicates that the electrochemical impedance spectroscopy can be taken as a nondestructive testing measurement to real-time monitor the cement hydration process of cement-based materials. ... To understand the influence of the accelerator on pore structure of the paste systems, the formation factor is evaluated following the Archies law (Eq. (2)) [58][59] [60] . ... ... The σ eff is bulk conductivity, σ o is pore solution conductivity, B is a constant and considered as 1 [60] , φ is pore volume fraction, and m is Archie's exponent, which is a function of system's geometry. The formation factor represents the condition of pore structure and tortuosity in the bulk system [48,58]. ... Criticality of microstructural evolution at an early age on the buildability of an accelerated 3D printable concrete Article Aug 2022 CONSTR BUILD MATER <here is a image 9b5bac676745df17-f31a6eff98d51ce8> Shantanu Bhattacherjee <here is a image bc6e011be23891fa-6e161a802f644d25> Smrati Jain <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam The buildability of a 3D printed concrete structure is influenced by the layer compression during printing, which in turn is governed by the rheological and early age mechanical properties of the mix. The study aims at understanding the effect of alkali-free aluminium sulphate-based accelerator on the microstructural evolution, which affects the macrostructural behaviour governing buildability of the structure. The effect of accelerator on hydration, phase composition, location and arrangement of ettringite, and porosity evolution of two paste systems with and without viscosity modifying admixture and superplasticizer is observed at initial hours. Further, fresh properties, yield stress evolution, and mechanical strength at early and later ages of the printable mix is evaluated. The squeeze flow test, which simulates the physical process of printing, is performed to understand the rheological characteristics of the mix under compressive loading in the initial hours. It is concluded that the precipitation of ettringite in large quantities at the initial hours controls the rheological and mechanical behaviour of the mix. The total ettringite formed increases for accelerated paste system at 30 min compared to the paste without accelerator. The ettringite is located at surfaces of clinker and interstitial spaces, with the crystals interlocked physically with each other. The squeeze flow test further suggests that the flow hardening increases tremendously on increasing the accelerator dosage due to an increase in solid volume fraction of ettringite and the resultant physical interlocking. Finally, a theoretical model is proposed considering the significance of ettringite interlocking and microstructure densification under compression on the plastic flow hardening for accelerated mixes. ... Some hydration products cover the surface of the cement particles and hinder further hydration, whereas the ion concentration in the pore water is in dynamic equilibrium with the dissolution and hydration reactions. Then, in the acceleration period of the hydration reaction, pore water is consumed to form large amounts of hydration products, and this process causes a change in the microstructure and pore characteristics of the cement slurry [4,18, 54, 55]. The electrical conductivity of the cement slurry subsequently begins to decrease rapidly. ... ... The electrical conductivity of the cement slurry increased with an increase in evaporable water content, which is similar to the relationship between porosity and electrical conductivity reported by Sanish et al. [54] . Combining with the results of Fig. 6 and Table 4, the analysis of the relationship between the free water content and the electrical conductivity of the cement slurry revealed a linear relationship (see Fig. 20 (c)), which indicated that the free water content was a key factor influencing the electrical conductivity of the cement slurry. ... Effects of microstructure and pore water on electrical conductivity of cement slurry during early hydration Article Sep 2019 COMPOS PART B-ENG <here is a image fcb66305a4f729ab-53cf310e5b3eb84f> Kaiqiang Liu Xiaowei Cheng Jingxue Li Chunmei Zhang In this study, inductance-type electrical conductivity measurement (IECM) was performed to study the electrical conductivity of different formulations of cement slurry. Low-field nuclear magnetic resonance, in situ X-ray micro-tomography, and environmental scanning electron microscopy were used to investigate the effects of the microstructure, pore characteristics, and pore water of cement slurry on its electrical conductivity. Experimental results showed that the pore characteristics and ion concentration governed the initial electrical conductivity of fresh cement slurries. The sedimentation stability of fresh cement slurries increased the number of interconnected pores, and the initial electrical conductivity increased from 16.8 mS/cm to 19.4 mS/cm. When the CaCl2 accelerator content was 2 wt%, the initial electrical conductivity of the fresh cement slurry increased to 26.9 mS/cm, while the electrical conductivity rapidly decreased with the formation of hydration products. With increasing hydration time, large amounts of hydration products were formed in the pores, which changed the microstructure and pores of the cement slurry from connected large volume pores to many small volume pores and subsequently changed the distribution of pore water. The experimental results revealed that the connected porosity of the cement slurry was proportional to the electrical conductivity. An exponential relationship was found between the electrical conductivity and the evaporable water content, whereas a linear relationship was found between the electrical conductivity and the free water content. ... In recent years, nondestructive techniques for indirect assessment of concrete transport properties and durability by measuring electrical conductivity (σ) or resistivity (ρ) have gained popularity [1]. e measurements are taken by several available methods based on uniaxial or bulk resistivity, surface resistivity, electrical impedance spectroscopy, and noncontact resistivity measurements based on the transformer principle [1][2][3][4] [5] . Additionally, embedded sensors for real-time ρ monitoring were evaluated in past studies [6][7][8]. ... ... Additionally, embedded sensors for real-time ρ monitoring were evaluated in past studies [6][7][8]. Since electrical current in the concrete is primarily conducted through the pore solution in the pore system, electrical measurements can be used to indirectly evaluate pore system and transport properties of concrete [5, 6,9,10]. Relatively high ρ (low σ) is a characteristic of concrete with dense microstructure, low porosity, and poorly interconnected pores. ... Evaluation of Methods to Correct the Effect of Temperature on Electrical Conductivity of Mortar Article Full-text available Dec 2018 <here is a image bedc8434666f0d0a-f3b29968c3f6b35d> Milena Rangelov <here is a image 4c2b6d099c12b17c-b93bfcb49e41b3ef> Somayeh Nassiri Nondestructive methods to obtain the electrical conductivity ( σ ) or resistivity ( ρ ) of concrete are gaining popularity for durability evaluation. However, these methods are susceptible to the effects of curing and conditioning, primarily temperature and degree of saturation. Before σ of concrete at varied temperatures can be used for durability assessment, appropriate corrections must be made to account for the effect of temperature ( T ). In this study, two existing and one new temperature correction methods were evaluated for 12 mortar mixtures varying in water-to-cementitious material ratio (w/cm) and the content and types of supplementary cementitious materials (SCM). Mortar specimens instrumented with embedded sensors were cured in sealed conditions for 11–13 months. After this period, the sealed specimens were subjected to stepwise temperature change in 5–50°C range while σ was recorded using the embedded sensors. Linear, bilinear, and Arrhenius temperature correction (LTC, BLTC, and ATC, respectively) were fitted to the obtained σ - T datasets and were evaluated for fitness. LTC provided an acceptable fit to the σ - T data ( R² > 0.81) but was found the most suitable in 5–30°C temperature range. BLTC was defined as a combination of two distinct LTC below and above the reference temperature at 23°C and had a better fit to the data ( R² > 0.96). Lastly, ATC showed the best fit among the tested methods ( R² > 0.98) and was found applicable for the full tested temperature range. Comparison of correction coefficients among the mixtures indicated that increase in w/cm results in less sensitivity of σ to temperature. Mixtures with SCM generally exhibit higher temperature sensitivity compared to the corresponding plain mixture. Since the variations in correction coefficients were not substantial (less 18% variation among 10 of 12 mixtures), a single value of activation energy of conduction ( Ec ) at 32 kJ/mol was identified as the general recommendation for all the tested mixtures. ... Total porosity is used in Eq. (6) because S n transforms FF for non-saturated concrete (left-hand side of Eq. (6)) into the respective FF for a fully saturated mixture [21]. In studies of rock formations and pervious concrete, m was defined as a constant ranging from 1.5 to 4 in value disregarding the develop- ment of m with time and dependence on mixture design [20,[22][23] [24] [25]. The results of a study by Nokken and Hooton (2008) also sug- gested a range from 1.34 to 4.73 for m in hardened concrete, rather than a time-dependent relation to define the physical meaning of m [26]. ... ... Based on the results in Fig. 8, simple regression models for m as a function of time and mixture design parameters were developed and are presented in Eq. (30). Current literature, which commonly used constant m-values regardless of elapsed curing time or mix- ture design [24, 25]. The results of this study and the regression models below allow more appropriate implementation of Archie's law for cement composites in the future. ... Empirical time-dependent tortuosity relations for hydrating mortar mixtures based on modified Archie's law Article Full-text available May 2018 CONSTR BUILD MATER <here is a image bedc8434666f0d0a-f3b29968c3f6b35d> Milena Rangelov <here is a image 4c2b6d099c12b17c-b93bfcb49e41b3ef> Somayeh Nassiri Using sensor-based electrical data, empirical time-dependent tortuosity (τ) formulas as functions of pore size distribution (PSD) parameters were developed for 12 hydrating mortar mixtures. The inverse of the formation factor (FF) was used to obtain Archie's exponent (m), which is an indicator of tortuosity. PSD parameters were inferred from theoretical water vapor sorption isotherms. The results clarify the effects of curing time and mixture design on FF and tortuosity. Additionally, it is shown that m correlates with pore system refinement and increase in disparity of pore sizes, suggesting the usefulness of this parameter in describing pore system's intricacy. ... As a dynamic porous composite material, cementitious materials consist mainly of solid (aggregates, cement clinker, and hydration products), liquid, solid-liquid interface, and gas phases. The electrical conductivity of the solid and gas phases in cementitious materials is also considered to be several orders of magnitude smaller than that of the liquid phase [67] . According to the literature, a typical cement paste contains 20 % to 60 % free water [68]. ... Freeze-thaw damage assessment of engineered cementitious composites using the electrochemical impedance spectroscopy method Article Full-text available Apr 2023 Mater Des Ru Bai Shuguang Liu <here is a image 74f8d6f05ae43327-c0eba3ac6ab89915> Liqiang Yin Lin Li The mechanical properties of engineered cementitious composites (ECC) in service in cold regions can be significantly degraded by periodic freezing and thawing. In this work, the damage degree of freeze–thaw of ECC was systematically assessed by using the electrochemical impedance spectroscopy (EIS) technique. In addition, Nuclear Magnetic Resonance (NMR) Relaxometry measurements were also performed to obtain pore structure parameters, and the uniaxial tensile tests were also carried out to analyse the tensile performance after freeze–thaw cycles. From the acquired results, it was demonstrated that the EIS behaviour of ECC varied with the freeze–thaw cycles. The diameter of the Nyquist curve in high-frequency was gradually reduced by increasing the freeze–thaw cycles. Furthermore, the volume resistance of ECC after freeze–thaw gradually decreased with the increase in the number of freeze–thaw cycles. The simplified microstructure and conductive paths were used to describe the freeze–thaw damage mechanism of ECC. An equivalent circuit model of ECC exposed to freeze–thaw cycles was proposed, and the parameters of the equivalent circuit model were thoroughly analysed. The experimental findings clearly indicate that the EIS method is an appropriate technique for evaluating the damage degree of freeze–thaw of ECC. ... This conductivity technique has already proved to be pertinent in applications such as assessing body fat, total body water, and fat-free mass in clinical research (Kyle et al., 2004a(Kyle et al., , 2004b, testing the hypothesis of the lunar mantle and the Earth's mantle (Laumonier et al., 2017;A. Pommier et al., 2015;Anne Pommier et al., 2015), monitoring the microstructural evolution in cement materials (Sanish et al., 2013; Tang et al., 2016), and monitoring crack growth or damage state in ceramic matrix composites (Mansour et al., 2015;Morscher et al., 2014;Simon et al., 2017). The conductivity technique is not only suitable for laboratory studies, but also for developing industrial online monitoring techniques due to its simplicity and relatively inexpensive nature. ... In-situ electrical conductivity monitoring of slag solidification during continuous cooling for slag recycling Article Mar 2023 Bart Blanpain <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Ling Zhang Muxing Guo <here is a image 0871ccca40c00f9a-c8cb4a377d094267> Annelies Malfliet Large volumes of steel slag are produced annually, leading to significant environmental protection and sustainable development issues. An online technology to monitor the solidification process of steel slag can assist in obtaining the right mineralogy to valorize these slags or render them harmless. For this purpose, we investigated the electrical properties and microstructural relationships of a CaO-Al2O3-SiO2-MgO (CASM) slag during cooling using an innovative setup. The electrical impedance was determined over the frequency range of 20 Hz to 300 kHz at two cooling rates, and the solidification behaviour was observed simultaneously by confocal scanning laser microscopy (CSLM). Four zones can be distinguished in the conductivity-temperature curves for the slag cooled at 10 °C/min, whereas only two distinct zones are visible at 100 °C/min. The liquid fraction of the slag has a significant impact on the slag conductivity during cooling. The electrical conductivity is, therefore, an accurate indicator of the solidification degree. Different theoretical and empirical models were evaluated on their ability to relate the bulk conductivity of the slag to the liquid fraction. The empirical Archie's model proved to be the most suitable model for relating the bulk conductivity of the slag to the liquid fraction. In-situ electrical conductivity measurements during cooling can provide an online assessment of the slag solidification process, including indicating the appearance of solid precipitates, monitoring the growth of crystals, indicating complete solidification when no liquid phase remains, and indicating the cooling rate. ... The appropriate conductivity and relative dielectric constants are allocated to the aggregate, rubber, and mortar in the numerical simulation of the mesoscale model of the rubberized concrete, as shown in Table 1, respectively. This provides to compute and assess the internal potential distribution [46][47][48] [49] [50][51]. ... Mesoscale research on electric potential of rubberized concrete affected by rubber geometry Jul 2022 CONSTR BUILD MATER <here is a image 71530ec70d0d48e2-51a8d84e18169f26> Hao Jin <here is a image ffefb32f4f002a18-3a0095eae20fc05d> Donghao Yin <here is a image a4118b7410a6aa99-5ef744655a21011e> Shuo Yu Rubberized concrete is a multiphase composite material that is made up of aggregate, rubber, and mortar. The applied voltage is often considered as an influencing factor when studying its properties, such as chlorine ion impermeability and resistivity. However, the electric potential distribution inside the rubberized concrete under applied voltage conditions needs to be studied in depth. In this paper, mesoscale research was conducted on the effect of the rubber geometry on the electric potential of rubberized concrete. The results reveal three points. First, The inclusion of rubber has a certain attenuation or blocking impact on overall and local potential propagation, and the effect varies depending on the content, shape, and size of the rubber. Second, when the rubber contents and particle heights are equal, cube rubber outperforms octahedron and spherical rubber in terms of the potential barrier effect. Third, for the same rubber content, the smallest rubber size provides the best barrier effect of electric potential. The potential difference decreases as the rubber size increases, although the law is not fixed. ... Several techniques such as, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) were used by several researchers to investigate the hydration and hardening mechanisms (Shen and Liu 2019; Sanish, Neithalath, and Santhanam 2013; Liu et al., 2021). It is well recognised that the aforesaid approaches may not be able to provide continuous monitoring of hydration process data. ... A new approach to investigate the hydration process and the effect of algae powder on the strength properties of cement paste Full-text available May 2022 Meriem Chahbi <here is a image c3eefbd021bf569e-36bd2a3c1da7d0ce> Abdelhadi Mortadi <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Reddad el moznine <here is a image ce52d9c6522c1c4c-917b4200e22ef9dc> Halima Rchid Sargassum muticum, a Japanese brown seaweed, has recently invaded various shorelines throughout the world, including the Atlantic coast of Morocco, where it has developed significant populations. We report on the use of these algae as a partial replacement of Portland cement in cement paste as part of a long-term plan to combat this invasive seaweed. With a fixed water to cement ratio (W/C = 0.45), algae powder is added at 5%, 10%, 15% and 20% from the cement content in producing cement pastes. The hydration process was assessed using impedance spectroscopy throughout the setting period from 10 min to 28 days. Compressive strength tests were performed at 7, 14 and 28 days. An equivalent circuit model has been proposed. The results showed that the various strength properties of the cement paste increased or decreased with addition of algae powder. The compressive strength tends to increase with the addition of algae powder. This study shows that 10% addition of algae powder to cement paste showed an increase in compressive strength and when the addition increased to 20% the properties started to decrease. ... Many studies were conducted using electrical resistivity measurements to investigate the effect of w/c ratio, and they suggested that electrical resistivity can be used as method to estimate porosity, and monitor the cement hydration process [24,25]. Literature is so rich with many studies suggesting that electrical resistivity follows the microstructural development of material through cement-water reaction [26, 27, 28,29,30]. Resistivity mainly starts increasing with hydrates expansion due to capillary porosity dis-connectivity. ... Water to Cement Ratio Effect on Cement Paste Microstructural Development by Electrical Resistivity Measurements and Computer Illustration Full-text available Jul 2021 <here is a image 30ab03f094dc1ecc-dcafc1b5e6f5a1db> Farqad .Y Alsaffar Wei Xiaosheng The effect of the water-to-cement ratio on cement paste microstructural development was investigated using electrical resistivity measurements and computer modelling. Three cement pastes with 0.3, 0.35, and 0.4 w/c ratios were used in this study. The electrical resistivity measurements curve as a function of time for the three pastes shows that the lower the w/c ratio the higher the electrical resistivity through the whole time. The effect of overall porosity and initial distances between particles significantly influence the measurements. Although low w/c ratio paste has low liquid electrical resistivity comparing to other samples, still has high bulk electrical at all ages. The influence of particles proximity due to lower water amount was interpreted through monitoring the electrical resistivity development and computer illustration. The demonstration provides more explanation about the importance of water-to-cement ratio. Through the conducted experiments, empirical correlations were developed regard predicting compressive strength and autogenous shrinkage of cement pastes when all parameters are related to porosity. Setting time was also predicted by monitoring the critical points on the electrical resistivity curve. Scanning electron microscopy (SEM) was employed to elucidate the effect of the w/c ratio on the morphology of hydration products, and it was observed that high w/c ratio paste has more fibers per unite area comparing to lower w/c ratio pastes due to the long distances between hydrates for expanding. ... The former supposes that the time corresponding to the minimum value point and a turning point on the resistivity development curve is linearly related to the initial and final setting time; while the latter considers that the first peak value of the resistivity differential curve is the turning point of cement paste hardening, which is related to the final setting time of cement paste. Some studies [15, 16] proposed using a derivative of the conductivity-time curve to predict the setting time of concrete. They found that the initial setting time corresponds to the highest peak of the conductivity differential curve. ... Prediction Model of Concrete Initial Setting Time Based on Stepwise Regression Analysis Full-text available Jun 2021 <here is a image 25031df4b6477490-0237b9d41a03bcc3> Weijia Liu <here is a image d910a46b6f8726d8-3939b9478c9e2d03> Niu xu jing Ning Yang <here is a image efd4d60131f05fc6-59e9fd871a8d7709> Yu Hu Mass concrete is usually poured in layers. To ensure the interlayer bonding quality of concrete, the lower layer should be kept in a plastic state before the upper layer is added. Ultimately, it will lead to the prediction of concrete setting time as a critical task in concrete pouring. In this experiment, the setting time of concrete in laboratory and field environments was investigated. The equivalent age of concrete at the initial setting was also analyzed based on the maturity theory. Meanwhile, factors affecting the setting time in the field environment were studied by means of multiple stepwise regression analysis. Besides, the interlayer splitting tensile strength of concrete subjected to different temperatures and wind speeds was determined. The results of laboratory tests show that both setting time and interlayer splitting tensile strength of concrete decrease significantly with the increase of air temperature and wind speed. In addition, the equivalent age of concrete at initial setting remains the same when subjected to different temperatures, while it decreases obviously with the increase of wind speed. In the field environment, the equivalent age of concrete at initial setting is greatly different, which is related to the variability of relative humidity and wind speed. The average air temperature and maximum wind speed are the main factors affecting the initial setting time of concrete. Furthermore, a prediction model is established based on the stepwise regression analysis results, which can predict the actual setting state in real-time, and hence controlling the interlayer bonding quality of dam concrete. ... Hence, the proposed fitting models are suitable for LFC density and compressive strength computation without the constraints from the Jonscher model's limitation. The findings matched well with, 26, 27 where the electrical nondestructive method is a useful parameter used to monitor the microstructural features of cement-based materials. The use of electrical response shows that the proposed method is able to predict the compressive strength and density of the LFC. ... Determination of density and compressive strength of lightweight foamed concrete using frequency domain approach Article Full-text available Aug 2021 MICROW OPT TECHN LET <here is a image f2fcf5112c1b9344-c260a09e5ad53394> Lee Kim Yee <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Phua Yeong Nan <here is a image 90c060043cd85b0c-1c1ddd2dcb7c1a38> Siong Kang Lim <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Cheng Ee Meng A fast and nondestructive method for predicting density and compressive strength of Lightweight Foamed Concrete (LFC) is proposed. The measurement is carried out using a frequency domain approach to determine the Lightweight Foamed Concrete dielectric constant from a frequency range of 150 kHz to 8 GHz. The relations of the dielectric, density, and compressive strength of lightweight foamed concrete has been investigated, and the correlation models have been formulated using Jonscher fitting model. The results show a close estimation with the relative error controlled within 2.6% and 6.0% for the density and the compression strength computation, respectively. The proposed frequency domain method is shown to be effective and can be applied to determine the mechanical attributes as well as the electrical properties of the LFC simultaneously. ... The measurement cell is connected to a computer-controlled Solartron 1260 EIS impedance analyzer, which is used to collect impedance spectra of mortar cube specimens over a frequency range of 0.1 Hz to 1 MHz with an applied AC voltage of 250 mV [55] [56]. Impedance spectrum of empty measurement cell was collected and subtracted from mortar cube spectrum results to correct the sponge material effect. ... Evaluating the impact of curing temperature in delayed ettringite formation using electrochemical impedance spectroscopy Full-text available Feb 2021 CONSTR BUILD MATER <here is a image 667c8030e50fe504-c5cae87bff0f7942> Yogesh Kumar Ramu <here is a image 66628851a9601197-492cd7d8e3fbfd35> Vute Sirivivatnanon <here is a image 9ba904d5eaa327ca-921b026abb023c5f> Paul Stephen Thomas <here is a image 91d6680849d5ace6-73f24277e6711820> Kirk Vessalas Delayed ettringite formation (DEF) could be deleterious and reduce the serviceability of structures built with precast concrete elements subjected to a short-term heat curing regime. Over 3 decades of research many characterization techniques have been developed to study the impact of DEF in cementitious systems. This paper attempts to assess the physical alterations due to DEF in cementitious systems using electrochemical impedance spectroscopy (EIS). Mortar specimens were made with two different cements exposed to different curing regimes and prone to DEF attack. At later age of 500 days after the significant expansion due to DEF, impedance arcs of mortar specimens were collected. Electrical resistivities owing to continuously connected pores and discontinuously connected pores were calculated with the impedance values. Electrical resistivities were compared with linear expansion and mass change of mortars. Results show a significant reduction in impedance with an increase in curing temperature due to physical damage and electrical resistivity was lowest for mortars with high DEF expansion. Precipitation of heterogenous DEF in confined microstructure capable of generating expansion further contributes to the lowering the electrical resistivity. This research shows EIS can be used to monitor the physical damage in the microstructure of the cementitious system to study deterioration mechanisms such as DEF. ... Figure 4-15 shows a schematic representation of the conductive path in concrete systems. Thus, conductivity measurements are sensitive to the microstructural changes occurring in the system and are often used to study early hydration changes, setting and an indication of pozzolanicity during the hydration process (Christensen et al. 1994;McCarter et al. 2000;Nokken and Hooton 2007; Sanish et al. 2013; Sant et al. 2009;Scuderi et al. 1991). ... COMPOSITE CEMENTS WITH LIMESTONE ADDITIONS: MICROSTRUCTURE AND TRANSPORT PROPERTIES Thesis Sep 2020 <here is a image 2db86de176e942d6-7441e4030f977ade> Yuvaraj Dhandapani Two-component systems (i.e., Clinker + supplementary cementing material or SCM) compose a major share of cementitious materials in the current scenario. A ternary blended system, with co-substitution of limestone, has the potential to complement the SCMs’ reaction and improve substitution levels of portland clinker. The synergistic interactions between the three components, i.e., clinker-limestone-aluminosilicates, has led to the adoption of finely grounded limestone -aluminosilicate combination as a cement substitute. However, there is a lack of understanding on the complexity brought into such ternary binder systems due to the unique physicochemical contribution of limestone in the hydrating cement matrix, particularly from the viewpoint of describing the physical structure development and its influence on concrete durability parameters. With this background, the current study was proposed to delineate the interaction mechanisms in composite binders by investigating the role of ternary binder systems on binder chemistry, phase assemblage, physical structure development and durability potential of concrete systems. ... On one hand, electrical conductivity or resistivity was often used for monitoring the evolution of porosity in cement pastes and concretes using 28-day experimental measurement [12] [13]. On the other hand, relative dielectric constant at various frequency ranges were studied as a hydration indicator whereas their properties in J o u r n a l P r e -p r o o f 3 correlations with compressive strength were established [14][15][16]. ... Prediction of Concrete Compressive Strength Based on Early-Age Effective Conductivity Measurement <here is a image d84c7d325f798177-e2fd7967839e2ed5> Kwok Chung <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Lingling Wang <here is a image 6e7aebd72fec5029-92e7f050ccd9cefa> Mohamed Ghannam <here is a image d020f00bc64b0007-26eb12da8ea27bf4> Jianlin Luo Prediction of compressive strength of concrete at early ages has attracted a lot of attention, but several defects still remain in previous researches using nondestructive techniques such as the use of electrical surface resistivity. A true prediction model for the 28-day compressive strength of concrete via effective conductivity at microwave frequency is presented for the first time in this paper. The open-ended coaxial probe was used to undertake the loss factor measurement, in the frequency range of 0.1 GHz to 10 GHz, of ordinary concrete with four water-cement ratios (0.40, 0.45, 0.49, 0.60) at first seven days. Traditional destructive test was used to obtain the concrete compressive strength at 3, 7, 14, and 28 days of curing. The reduction of conductivity versus curing age was modeled by using exponential decay function whereas the development of compressive strength curing age was modeled by using a nonlinear raising function. By taking curing age as an intermediate variable, a linear prediction model as a function of effective conductivity was developed. A minimum effective conductivity was discovered where the frequency of about 3 GHz was considered as the dominated loss mechanism transits from ionic conduction loss to dipole polarization loss. Our results demonstrated that the 28-day compressive strength of concrete having close water-cement ratios can be accurately predicted using effective conductivity. Moreover, the magnitude of the slope of strength-conductivity curves could give an indication on the grade of standard concrete during early ages. The model was evaluated by using data from open literature with DC conductivity and the accuracy was obtained with about 10% error in underestimation. ... Accelerators are soluble electrolytes whose ions may affect the charge of particles. Conductivity of cement paste depends on the types of ions and their concentrations [28] . The conductivity, pH and zeta potential of cement paste increased with time. ... Structural build-up, hydration and strength development of cement-based materials with accelerators CONSTR BUILD MATER Dajun Zhou Hai Huang <here is a image 652e492331305e3d-2a24370dcf562cef> Hao Yao <here is a image da715728fb66359d-80f1eb03dcdf836f> Qiang Yuan Fast structural build-up and strength development of fresh/hardened cement-based materials are always desired in many scenarios, where accelerators are often used. In this paper, the hydration, structural build-up and strength development of cement pates with different accelerators were investigated. The growth of static yield stress was used to characterize the structural build-up of fresh cement paste with 1% nano silica. Calorimetric curves, zeta potential and conductivity of pastes and strength development of mortars were also measured. Results showed that accelerators promote the reaction of C3S or C3A at different stages and in different mechanisms. Mechanical efficiency of C-S-H determined by growth of elastic modulus or yield stress over cumulated hydration heat may give wrong ranking on the efficiency of C-S-H. Apart from formation of C-S-H gel, structural build-up may originate from other sources. ... Impedance response was captured using a Solartron 1260 instrument between a frequency range of 0.1 Hz-1 MHz with an AC voltage of 250 mV. The intersection point of the bulk and electrode arcs from the Nyquist plot was considered as the resistance of the bulk cementitious systems to calculate electrical conductivity from paste systems, as reported in previous studies [17, 28] . ... Investigation on the microstructure-related characteristics to elucidate performance of composite cement with limestone-calcined clay combination Mar 2020 CEMENT CONCRETE RES <here is a image 2db86de176e942d6-7441e4030f977ade> Yuvaraj Dhandapani <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam This paper discusses the role of physical structure alterations on three binder types: plain portland cement, fly ash-based binder and calcined clay-limestone binder. The kinetics of physical structure development and the relevance in transport properties were distinguished using an interlinked parameter in concrete and paste. A systematic experimental investigation was carried out on a range of critical parameters such as strength development, resistivity development, transport characteristics and the time-dependent change in transport parameter. The role of microstructure in terms of the chemical composition of C-A-S-H and its physical states in the different systems is identified as the critical factor governing the development of microstructure. Chloride resistance was assessed by chloride migration experiments for a period of 4 years. The durability behaviours of the concrete with various binder were generalised using pore network parameters such as formation factor and tortuosity. A sensitivity analysis was used to dissect the contribution of the pore solution dilution and pore connectivity to the change in the pore network parameter. Based on the rise in macroscopic physical characteristics (i.e., formation factor here), a two-fold structure development mechanism to conceptualise microstructural evolution in cement composites is presented. Initially, capillary pore space reduces to a critical size range (i.e., 10-30 nm), which is followed by the densification of the physical state of the microstructure. At the point of densification, the pores become largely disconnected which leads to a dramatic increase in the formation factor. The binding matrix in calcined clay concretes reaches the critical pore size at an early age which leads to early densification of capillary pore space region in comparison to fly ash concretes. ... Experimental result shows that with increase in size the impedance response has better resolution to determine the IST and FST values. However, the experiment should be carried outin details with large number of sensors having different sizes However, the variation did not exceed the tolerance value as reported in [49] . ... Fringing Field Impedance Sensor for Hydration Monitoring and Setting Time Determination of Concrete Material May 2019 IEEE T INSTRUM MEAS <here is a image 43d4fd3422de85cd-7ebad6a82d91d9ee> Kazi Jabed Akram <here is a image 969432077710d9c3-dc9fc08289f01226> Akil Ahmed <here is a image 1c68dd6e89bab0f0-c5391cada2a9a89c> Tarikul Islam This work deals with the development of a simple, low cost, fringing field impedance sensor suitable for early age hydration monitoring and setting time determination of hydraulic cement paste. An impedance technique using double sided copper cladded PCB substrate as a sensor is proposed. Different batches of sensors with different probe sizes and substrate thickness have been fabricated and tested for hydraulic cement paste. The cement samples having different water to cement ratios (w:c) by weight are considered to study the effectiveness of the sensor. The purpose of this study is to develop a simple yet effective measurement system to determine the change in impedance of the cement paste with hydration time and thus to correlate the impedance change to determine the critical initial and final setting times. Initial and final hydration times of different cement samples are compared with the results of commercial Vicat apparatus. The values of the setting times closely match the Vicat values (the average value at 100 kHz differs by 4 m). So, the proposed sensor-based system can be used in place of conventional Vicat apparatus to determine the hydration condition. The output of the sensor can be electronically processed further to develop a prototype system. ... Despite the presence of numerous obstacles in the growth of both indexes shown in Fig. 5 such that the solution is supersaturated with respect to calcium hydroxide, the overall hydration trend is promising. The beginning of the acceleration period (T II ) is always associated with the precipitation of Portlandite, which has been recently confirmed by coupling the isothermal calorimetry with the electrical-conductivity measurements [32] . During the early acceleration period (T II to T IN to T FN ), the impedance change rate first increased and then significantly decreased; meanwhile, the hydration heat increased until the deceleration period. ... Development of mathematical models for predicting the compressive strength and hydration process using the EIS impedance of cementitious materials May 2019 CONSTR BUILD MATER <here is a image 9eb7efc4d08d5d61-09f2e0dd49fd0571> Lin Chi Zheng Wang <here is a image 40cffecec132da49-df2247f16c3bfa9f> Shuang Lu <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Yan Yao ... Impedance response was captured using a Solartron 1260 instrument between a frequency range of 0.1 Hz-1 MHz with an AC voltage of 250 mV. The intersection point of the bulk and electrode arcs from the Nyquist plot was considered as the resistance of the bulk cementitious systems to calculate electrical conductivity from paste systems, as reported in previous studies [17, 28] . ... CHARACTERISATION OF MICROSTRUCTURE IN LIMESTONE CALCINED CLAY CEMENTITIOUS SYSTEMS Conference Paper Mar 2019 <here is a image 2db86de176e942d6-7441e4030f977ade> Yuvaraj Dhandapani <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam Recent studies on the binder and concrete properties of limestone calcined clay cement (LC3) have shown good potential for producing high-performance, low clinker cement with a combination of limestone and calcined clay [2]. The greater resistance to ionic ingress, more specifically chloride ingress and sulphates, was found to be strongly influenced by the reduced pore sizes due to higher reactivity of calcined clay present in LC3 systems [2]. Even lower grade calcined clay results in an early refinement of pore structure due to highly pozzolanic nature of the amorphous phase [3] and dense microstructure formed in such systems [4]. This alteration in the physical structure is related to the nature and chemistry of hydration products and the variation in the composition of C(A)SH formed in these systems discussed in this paper. ... where r wat ¼ 10 À5 S=m, corresponding to the water conductivity as found in [45]. value is of the same order of magnitude as the values found in the literature, although the literature values obviously depend on cement type and water to cement ratios [26, 41, 42]. In order to determine the factor n, which is assumed to be constant for both carbonated and non-carbonated slabs, the data obtained on non-carbonated slabs are used. ... Combination of the Wenner resistivimeter and Torrent permeameter methods for assessing carbonation depth and saturation level of concrete CONSTR BUILD MATER <here is a image 7ca05a2c38b62eed-1a899b4ba3d5eb0a> Stéphanie Bonnet <here is a image 3f42a5d702a31080-41f2acc52bd6e4eb> J. P. Balayssac Assessing carbonation depth is of great interest for the diagnosis of reinforced concrete structures because carbonation is one of the origins of steel corrosion. The assessment of carbonation depth is usually performed by a simple and reliable semi-destructive test consisting in spraying a colored indicator on a sample extracted from the structure. When the structure is large, this test must be reproduced many times if an assessment of the variability of carbonation depth is required. In this case, the extraction of multiple samples can be prohibitive from the technical and economic points of view. So, in this case, a non-destructive testing (NDT) method could be relevant. However, even when NDT methods can be used, there is a need to improve the interpretation of their results. In this study, the use of two usual NDT methods is proposed: resistivity measurement by a Wenner probe and surface permeability assessment by a Torrent permeameter. Both techniques are implemented on carbonated slabs having different carbonated depths and at different saturation degrees. The results show that the two techniques are sensitive to moisture and carbonation. For a given saturation level, resistivity increases when the carbonated depth increases and resistivity decreases when the saturation level increases. Torrent permeability decreases when the saturation degree and carbonated depth increase. Good repeatability is observed for resistivity measurements while a larger scatter is obtained for Torrent permeability. Empirical laws are built for the relationships between resistivity or permeability and saturation degree and carbonated depth. For resistivity, either a linear model between resistivity and saturation degree is used or Archie's law is adapted to take the effect of carbonation into account. Following the same idea, a linear law between Torrent permeability and saturation degree is adapted to the effect of carbonation. Because resistivity can be measured only if the saturation degree is rather high, i.e. if there is a continuity of the interstitial solution, and because permeability assessment is impossible if the concrete is fully saturated, these laws are limited to saturation degrees in the 40–83% range. Then, these laws are used to predict carbonation depth and saturation degree on a wall designed with the same concrete but stored in different conditions. The results show that resistivity and Torrent permeability can be used for the combined assessment of carbonation depth and saturation degree in laboratory conditions. ... It can be seen from Fig. 12 that among numerous models, Archie's Law and GEM model could be the best predictive models when applied to cement-based materials. Moreover, for a multiphase mixed system with different conductive phases, the multi-phase phenomenological model [61, 62] Fig. 7. "Ink-bottle" pore shapes of different pore size [37]. of these three models in correlating pore structure of cement-based materials to formation factor are discussed in this section. ... Determining porosity and pore network connectivity of cement-based materials by a modified non-contact electrical resistivity measurement: Experiment and theory ... Electrical conductivity is an effective parameter to monitor the microstructural features of cementbased materials. Non-invasive determination of the pore structure features, through the use of electrical response, was discussed in the paper [14] . Changes taking place in cementitious materials as a result of the hydration process were monitored based on the change in conductivity with time. ... Non-destructive control method of the state of objects operating long time The development of a diagnostic system is a complex engineering problem aimed at preventing damage caused by deteriorating properties of the materials and structures. This article presents the method of nondestructive testing of change control of concrete structures. The analysis of literature data shows that the overwhelming majority of studies are devoted to the change in electrical resistance of concrete in the collection of its strength, and practically no analogous studies regarding its aging are presented. Theoretical substantiation of the change in electrical concrete characteristics (conductivity, resistance) is given with a change in its strength. A work over-bridge in operation more than 30 years has been surveyed. The experimental part consisted in measuring the electrical potentials on the bridge structures, as well as in determining the strength of the material at the same points. Correlation dependence between the measured electric potential value and the strength of the samples taken from the bridge structures were obtained. ... Electrical methods have shown considerable potential to study cement reaction mechanisms like hydration, setting etc. [1] [2] . The use of Electrical methods allows a non-invasive and continuous assessment of the properties at various period of hydration. ... Electrical conductivity based evaluation of solid phase growth and microstructural evolution in multi-component cementitious systems ... The bulk resistance was taken as the intersection point of the bulk and electrode arcs from the Nyquist plot using an equivalent electrical circuit approach. The electrical conductivity of the system was calculated from the bulk resistance [18] . As the evaluation was non-destructive, the measurement was continued on the same specimen for 1000 h. ... Assessment of pore structure evolution in the limestone calcined clay cementitious system and its implications for performance Use of limestone and calcined clay together for clinker substitution makes an effective low clinker cement blend, which shows promising mechanical properties at early ages. The performance of these cementitious systems strongly depends on the pore structure, which is a dominant factor governing the durability characteristics because of its direct influence on the transport properties. The experimental study described in this paper on three different binder systems including Ordinary Portland Cement, Portland Pozzolana Cement-with 30% Type F Fly Ash (designated FA30)-and Limestone Calcined Clay Cement (LC 3) pastes shows that the LC 3 system attains greater refinement of the pore structure as early as 3 days, as seen from mercury intrusion porosimetry. Electrical measurements also reveal lower conductivity in the system, which suggests better resistance to ionic transport in the binder phase. The results of hydrate phase assemblage studied by X-ray diffraction also indicate that greater amount of hydrates contribute in a major way to the reduction in the (water-filled) porosity in all the systems. This change occurs at varying rates for the different systems due to the difference in hydration characteristics. The estimated permeability suggests that the LC 3 binder system attains much lower permeability compared to the ordinary Portland cement and FA30. A comparison of the formation factor shows distinct differences in the microstructural development and suggests a more durable binder with LC 3 cementitious system. Structural and dynamic characterization of two-component waterproof mortars by 1H NMR, FT-IR, mechanical and SEM investigations Evaluation of a Cross-Conductance Sensor for Cement Paste Hydration Monitoring and Setting Time Measurement <here is a image da8403c238c600ab-04869d7f0c6ed87c> Boby George <here is a image 43d4fd3422de85cd-7ebad6a82d91d9ee> Kazi Jabed Akram <here is a image 969432077710d9c3-dc9fc08289f01226> Akil Ahmed <here is a image 1c68dd6e89bab0f0-c5391cada2a9a89c> Tarikul Islam In this paper, a sensor for the monitoring of hydration and detection of initial and final setting timings of freshly mixed cement paste is presented. It is a new application of a cross-conductance sensor and possesses the advantages of the cross-conductance approach, i.e., the output is sensitive only to the conductivity of the medium other than the length of the electrode. The new sensor overcomes several limitations of the conventional Vicat Apparatus and other existing impedance-based schemes. The cross-conductance sensor cell is designed with the help of finite element analysis. The cell has four conductive electrodes separated by small gaps. It is fabricated using inexpensive but reliable materials. A simple measurement circuit is developed to obtain an output due to a change in the conductance of the sensor cell. The output of the sensor unit gives the conductance versus the time response of the sample. From the rate of change of this output, setting time of the cement paste samples are determined. The proposed sensor is simple to realize and it does not require calibration. The repeatability of the prototype sensor unit is found to be 1.5%. With the help of the fabricated sensor, the initial and the final setting timings of the cement paste samples are measured with an accuracy of ± 2%. Application of Sustainable Cement Alternatives in Concrete Considering sustainability as issue which means creating or maintaining changes in a balanced structure. Concrete is considered as the main constituent of the building industry and its main component is cement but cement releases almost 5 % of Co2 of the world wide emission. To use sustainability as the prominent resources in order to conserve future generation and to find the alternative materials of cement as it emits more Co2 while production. Cement can be replaced by many materials like fly ash, slag, silica fume, metakaolin, egg shell powder, rice husk ash etc. Cements alternative materials such as by-products are been researched upon. Factors Influencing the Electrical Properties of Ettringite Binders as Repair Materials Ettringite binders are increasingly being used as rapid-repair materials to extend the service-life of reinforced concrete where rebar corrosion has caused deterioration and spalling. The transport properties of these ettringite binders are important in assessing the longevity of such repairs. This study demonstrates how the electrical properties of such binders can be interpreted to obtain meaningful results related to the transport properties. In addition to the pore solution chemistry, the degree of saturation in calcium sulfoaluminate (CSA) cement was found to be an important factor in the electrical properties since ettringite binders can have greater self-desiccation at earlier ages than ordinary portland cement (OPC). After one day of hydration, the electrical properties suggested that the CSA containing mixtures had greater pore network connectivity than the OPC mixture and this can be attributed to the varying phase compositions. Thermodynamic modelling confirmed a high amount of ettringite formation in the CSA system and a lower capillary water and capillary porosity associated with this. The lower internal relative humidity of ettringite binders should be considered when using electrical measurements to evaluate their transport properties since it increases the total electrical resistivity. Features of Immittance Spectra as Performance Indicators for Cement-Based Concretes APPL SOFT COMPUT Jifeng Guo <here is a image baf61ec21497f1ac-1a2181be6b63fb6b> Lin Wang Kaipeng Fan <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Bo Yang Cement is the main building material in the construction industry. Its setting time directly affects the setting time and strength of concrete, which further affects construction schedule and building quality. However, traditional measurement technology not only has high labor intensity and high time consumption, but has a high technical requirement. Various human factors, such as insufficient operation, will result in great errors in the measurements. The accurate prediction of setting time enables manpower savings, avoids large errors caused by insufficient operation, and guides the production of high-performance cement. In this paper, an efficient model based on the broad learning system is proposed to predict the initial and final setting time. It is committed to directly predicting setting time from clinker composition and physical properties, which is of great significance to the optimization of clinker formula. The experimental results show that it can accurately predict the setting time and behave good generalization ability, which addresses the problem of labor intensity in measurement and saves many resources. In addition, the broad learning system can rapidly build a setting time prediction model with few errors, satisfying industrial demands for the rapid modeling of various specialty cements. A review on fractal footprint of cement-based materials POWDER TECHNOL <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Shengwen Tang <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Jiasheng Huang <here is a image ae347c91261ab71c-5dba316ee7f0c889> Lian Duan <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> E. Chen Cement-based materials are the most used construction and building materials in the world. This paper reviews the state-of-the-art of fractal geometry on cement-based materials. The porous structure of cement-based materials is extremely complicated and irregular, which is difficult to be described in terms of geometry. The fractal geometry is widely applied to investigate the pore structure of cement-based materials. In this work, the fractal characteristics of porous cement-based materials combined with the mathematical methods are discussed. Some direct and indirect methods for the evaluation of fractal dimensions are introduced, such as mercury intrusion porosimetry (MIP), nitrogen adsorption/desorption (NAD) and nuclear magnetic resonance (NMR). Some experimental results from different methods are compared to illustrate the advantages and disadvantages of these methods. Finally, correlations among fractal dimensions and properties of cement-based materials, such as permeability, diffusivity, strength and fluidity, are summarized. Structural Health Monitoring Using Impedance Sensor Conference Paper <here is a image 863f8b0b2e9e025a-0fea8d67194446b6> Zubair Hassan Zargar <here is a image 43d4fd3422de85cd-7ebad6a82d91d9ee> Kazi Jabed Akram <here is a image 1c68dd6e89bab0f0-c5391cada2a9a89c> Tarikul Islam <here is a image 969432077710d9c3-dc9fc08289f01226> Akil Ahmed Durability of glass fibre-reinforced polymer (GFRP) bars embedded in concrete under various environments. I: Experiments and analysis COMPOS STRUCT This paper presents the tensile strength of glass fibre-reinforced polymer (GFRP) reinforcing bars embedded in concrete that is exposed to tap water, saline solutions and ambient humidity under accelerated conditions. These conditions were used to simulate the effect of river water, seawater or atmospheric moisture on the GFRP bars. In addition, the electrical impedance method was employed to evaluate the water content at different depths of a concrete cover under a variety of ambient humidity conditions. The effects of seawater, water-to-cement ratio (W/C) of the concrete cover, environmental humidity and cover depth were studied. The results revealed that the GFRP bars embedded in concrete exhibited a substantial strength loss when exposed to tap water or high ambient humidity. The degradation of concrete-wrapped GFRP bars immersed in saline solution could be different. This discrepancy is due to the variation of the degradation mechanism after exposure to chloride ions and is related to the cover depth. The depth of the concrete cover also had an evident influence on the durability results of GFRP bars subjected to various humid environments. Furthermore, reducing the W/C of the concrete cover had negative effects on the GFRP reinforcing bars. The pore solution of cement-based materials: structure and dynamics of water and ions from molecular simulations PHYS CHEM CHEM PHYS <here is a image e98b75377abc673b-8c009abb0186b68f> Tulio Honorio <here is a image 3ac5520a263a6506-d5150f3bf09ff5af> Farid Benboudjema <here is a image 1937ee36a50dd27a-fa33d150fae83bfe> Thierry Bore <here is a image 18d28ea1bd11cf79-b526c83ca2c596b2> Eric Vourc'h Diffusion processes are crucial to the durability and confinement capacity of cement-based materials as well as property development. The liquid phase in cement-based materials is the pore solution, whose composition changes with age and is a function of the cement system composition. Water structure and dynamics are recognized to be affected by the presence of ions. Fundamental understanding of the physical processes underlying these changes can be critical in the elucidation of the physical origin of durability issues and in the development of new admixtures. Here, the structure and dynamics of water and ions present in pore solution are studied using molecular dynamics simulations. Self-diffusion coefficients are computed for bulk solutions mimicking the complex composition of pore solutions. Specific ion effects on water dynamics are observed and are interpreted in terms of water reorientation time. The composition dependency of ion dynamics explains the evolution of the ionic conductivity of the pore solutions. Design of the Cement-Hydration-Characteristics Measuring Device Yuanjiang Wang Zhenjiang Feng A Simple Method on Transformer Principle for Early Age Hydration Monitoring and Setting Time Determination of Concrete Materials IEEE SENS J <here is a image 43d4fd3422de85cd-7ebad6a82d91d9ee> Kazi Jabed Akram <here is a image 1c68dd6e89bab0f0-c5391cada2a9a89c> Tarikul Islam <here is a image 969432077710d9c3-dc9fc08289f01226> Akil Ahmed This work deals with the development of a lowcost measurement system based on transformer principle with lossy capacitive load for setting time determination of freshly mixed cement paste. The initial setting time is an important parameter to ensure transporting, placing and compacting of material to be utilized for the civil structure. Determination of final setting time helps in removal of a framework after appropriate strength gaining. The proposed method employs a test cell of Vicat instrument with two parallel metal plates. The cement paste behaves as a lossy dielectric kept in the test cell. To determine the setting times, the sample cell was connected to the secondary terminals of a transformer and the primary current was measured at a regular interval of time. The measured primary current was utilized to monitor the possible early age hydration and to determine the initial and final setting time of different mixture of the cement samples. Results are compared with the results of the conventional Vicat apparatus and are found to be effective for setting time determination. This proposed technique gives privilege for online hydration monitoring of the cement paste and can alternately be used in place of Vicat apparatus. Development of the Microstructure in LC3 Systems and Its Effect on Concrete Properties <here is a image 73050ca5d1776351-4417985af50b6949> K. Vignesh <here is a image 8dae13090a5f5c4e-88c786a629ba42db> Thangadurai Raja <here is a image 2db86de176e942d6-7441e4030f977ade> Yuvaraj Dhandapani <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam In this paper, the difference in the microstructure development of limestone calcined clay cement (LC3) in comparison with the ordinary Portland cement (OPC) and Portland pozzolan cement (represented as FA30) at different water-binder ratios is shown. The results from the studies suggest that a mere adoption of a lower water binder ratio to reduce the capillary pore space for filling by hydrates, ensures only a marginal improvement in the kinetics of microstructural development, as seen from conductivity evolution in OPC and FA30. Highly reactive pozzolans ensure a more rapid drop in conductivity due to low-density hydrates resulting in lower capillary porosity and densification of the hydrate matrix. BSE micrographs also show a more densified binder matrix with LC3, mainly due to high pozzolanicity of calcined clay resulting in CSH with lower C/S ratio with a hybrid hydrated phase assemblage compared to OPC and FA30. The impact of improved kinetics of LC3 binder reflects in better durability parameters at an early age in the different concretes made with LC3 binder. It is seen that the fly ash based systems (FA30) show a marked increase in the concrete resistivity up to an age of 1-year curing. The resultant effect of such microstructural development on the chloride resistance of concretes is also discussed. Service life estimation and life cycle assessment for portland cement, fly ash, and LC3 systems <here is a image 99d9e380f67d6047-ec3f39d948fb24c4> Radhakrishna Pillai <here is a image 5ea1d71b8a20e13d-197416d4084dd418> Ravindra Gettu <here is a image a0b0a796e38cb278-de348597b529c5b5> Anusha S. Basavaraj <here is a image 149ef4184b30b8a6-00e8c4ce6b318dfd> Manu Santhanam Portland cement, fly ash, and Limestone Calcined Clay Cement (LC3) are available in the market. Some of these materials claim enhanced service life for systems exposed to chlorides and/or carbondioxide. However, prior to their use, designers would like to have quantitative estimates of service life of concrete systems with such materials and the assessment of carbon footprint during their service life. Estimating service life of systems with these materials is very challenging because of the lack of knowledge on the key parameters influencing their service life. The key parameters include chloride diffusion coefficient (DCl), carbonation coefficient (KCO2), decay constant (m), and chloride threshold (Clth). This paper presents the quantitative estimates of these parameters. A total of nine concretes were designed. These included OPC, FA30 (30% flyash+70% OPC), and LC3 concretes with M30 and M50 strength grades (six concretes). Also, one mix each with OPC, FA30 and LC3 binders and same mixture proportion was included. The service life and life cycle of these nine concretes were assessed Study on hydration products by electrical resistivity for self-compacting concrete with silica fume and metakaolin This paper investigates the hydration products of self-compacting concrete (SCC) made with binary and ternary blends of silica fume(SF) and metakaolin(MK). X-ray diffraction (XRD) and thermogravimetric analysis (TGA) test were used to measure the hydration products of pastes corresponding to self-compacting concretes. For this purpose, the reference SCC mixture at constant water to cementitious material (W/CM) ratio of 0.45 and total cementitious material content of 450 kg/m³ was prepared. Other mixtures contained binary (92%PC + 8%SF, 80%PC + 20%MK) and ternary (72%PC + 8%SF + 20%MK) cementitious blends of metakaolin and silica fume. Four pastes corresponding to SCC mixtures with the same W/CM and cementitious materials were considered. Also, the electrical resistivity of SCC mixtures was measured to assess the relationship between electrical resistivity over time and calcium hydroxide (CH) content. The correlation coefficient of the relationship between Ca(OH)2 content and electrical resistivity was found to be 0.95 with positive slope for reference SCC and 0.97 with negative slope for samples containing pozzolans. It can be suggested to apply the electrical resistivity as a simple method to calculate CH content in cement paste.	https://www.researchgate.net/publication/270919592_Monitoring_the_evolution_of_material_structure_in_cement_pastes_and_concretes_using_electrical_property_measurements
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Materials \| Free Full-Text \| A Constitutive Model for Soft Clays Incorporating Elastic and Plastic Cross-Anisotropy Natural clays exhibit a significant degree of anisotropy in their fabric, which initially is derived from the shape of the clay platelets, deposition process and one-dimensional consolidation. Various authors have proposed anisotropic elastoplastic models involving an inclined yield surface to reproduce anisotropic behavior of plastic nature. This paper presents a novel constitutive model for soft structured clays that includes anisotropic behavior both of elastic and plastic nature. The new model incorporates stress-dependent cross-anisotropic elastic behavior within the yield surface using three independent elastic parameters because natural clays exhibit cross-anisotropic (or transversely isotropic) behavior after deposition and consolidation. Thus, the model only incorporates an additional variable with a clear physical meaning, namely the ratio between horizontal and vertical stiffnesses, which can be analytically obtained from conventional laboratory tests. The model does not consider evolution of elastic anisotropy, but laboratory results show that large strains are necessary to cause noticeable changes in elastic anisotropic behavior. The model is able to capture initial non-vertical effective stress paths for undrained triaxial tests and to predict deviatoric strains during isotropic loading or unloading. A Constitutive Model for Soft Clays Incorporating Elastic and Plastic Cross-Anisotropy by Jorge Castro 1,* and Nallathamby Sivasithamparam 2 1 Department of Ground Engineering and Materials Science, University of Cantabria, Avda. de los Castros s/n, 39005 Santander, Spain 2 Computational Geomechanics Division, Norwegian Geotechnical Institute (NGI), P.O. Box 3930, Ullevål Stadion, 0806 Oslo, Norway * Author to whom correspondence should be addressed. Materials 2017 , 10 (6), 584; https://doi.org/10.3390/ma10060584 Received: 6 April 2017 / Revised: 16 May 2017 / Accepted: 17 May 2017 / Published: 25 May 2017 Abstract : Natural clays exhibit a significant degree of anisotropy in their fabric, which initially is derived from the shape of the clay platelets, deposition process and one-dimensional consolidation. Various authors have proposed anisotropic elastoplastic models involving an inclined yield surface to reproduce anisotropic behavior of plastic nature. This paper presents a novel constitutive model for soft structured clays that includes anisotropic behavior both of elastic and plastic nature. The new model incorporates stress-dependent cross-anisotropic elastic behavior within the yield surface using three independent elastic parameters because natural clays exhibit cross-anisotropic (or transversely isotropic) behavior after deposition and consolidation. Thus, the model only incorporates an additional variable with a clear physical meaning, namely the ratio between horizontal and vertical stiffnesses, which can be analytically obtained from conventional laboratory tests. The model does not consider evolution of elastic anisotropy, but laboratory results show that large strains are necessary to cause noticeable changes in elastic anisotropic behavior. The model is able to capture initial non-vertical effective stress paths for undrained triaxial tests and to predict deviatoric strains during isotropic loading or unloading. Keywords: natural structured clays ; cross-anisotropy ; constitutive model ; anisotropy evolution ; stress-dependent stiffness ; triaxial tests Graphical Abstract 1. Introduction Extensive experimental testing of soils under different stress paths and conditions as well as the increase in computing power has led to the development of advanced constitutive models that reproduce more accurately the mechanical behavior of soils. Some of these advanced constitutive models incorporate plastic anisotropy using a rotated and distorted elliptical yield surface [ 2 , 3 , 4 , 5 ]. These models assume elastic behavior is isotropic, yet it is known most natural clays will exhibit anisotropy of elastic behavior [ 6 ]. The reasons to avoid incorporating elastic anisotropy are usually the following [ 3 ]: To keep the model simple. Because plastic deformations are likely to dominate for many problems of practical interest. Because, if the model attempts to account for the development or erasure of anisotropy with subsequent loading, that leads to changes in the initial cross-anisotropic behavior, potentially resulting in fully generalized elastic anisotropy, involving 21 independent elastic parameters, which is clearly not practical. Although the three above-mentioned reasons are sensible and justified, incorporating elastic cross-anisotropy to a model of this type is still useful because the model will better capture the elastic anisotropic behavior [ 7 ]. Besides, the development or erasure of elastic anisotropy requires important plastic strains; contrary to plastic anisotropy, which evolves at a much faster rate. For example, Mitchel [ 8 ] and Huekel and Tutumluer [ 9 ] showed that the rate of plastic anistropy demise is notably greater than that of elastic anisotropy after isotropic loading and unloading. The MELANIE model [ 7 ] is a constitutive model for soft soils that incorporates both elastic and plastic anisotropy and has been used to model real boundary value problems, namely several experimental embankments in Cubzac-les-Ponts (France) [ 7 ]. However, the MELANIE model does not consider two important features of soft clay behavior: evolution of plastic anisotropy with plastic straining and stress-dependent elastic stiffness. In this paper, an existing elastoplastic model that considers plastic anisotropy and its evolution with plastic straining, namely S-CLAY1S model [ 10 ], is extended to incorporate elastic cross-anisotropy, also known as transverse isotropy. To keep the model simple and for practical usage, only an additional parameter has been added to describe the elastic stress-dependent cross-anisotropy. Furthermore, evolution of elastic anisotropy is neglected. The additional elastic parameter has a clear physical meaning, namely the ratio between the horizontal and vertical stiffnesses, and it may be obtained analytically from a conventional triaxial test. The description of elastic cross-anisotropy is based on the three-parameter model proposed by Graham and Houlsby [ 6 ] for linear elasticity, but here, the stress-dependent response is also included. The paper first presents the S-CLAY1S model ( Section 2 ), as the proposed constitutive model is based on that model and the formulation of the plastic part is exactly the same. Next, the description of the elastic cross-anisotropy considered in the new constitutive model is developed ( Section 3 ). Validation of the model using laboratory tests available in the literature and examples of parameter determination are presented in Section 4 , and subsequently parametric studies are depicted in Section 5 . Finally, some conclusions are derived. The standard soil mechanics sign convention of compressive stresses and strains as positive is assumed, the prime notation is used to denote effective stresses ( σ ′ ), and engineering shear strains are adopted ( γ i j ). The stress and strain invariants are p ( σ ′ x x y / (1) y z 2 + τ x z 2 ) (2) ε v = ε x x + ε y y + ε z z (3) ε d = 1 3 2 [ ( ε x x − ε y y ) 2 + ( ε y y − ε z z − z γ x y 2 + γ x z 2 + γ y z 2 ) (4) which, for triaxial conditions, reduce to p ′ = ( σ ′ a + 2 σ ′ r ) / 3 (5) q = σ ′ a − σ ′ r (6) ε v = ε a + 2 ε r (7) ε d = 2 ( ε a − ε r ) / 3 (8) x , y , z denote rectangular Cartesian coordinates. For triaxial conditions, a and r subscripts indicate axial and radial principal directions, respectively. 2. S-CLAY1S Model S-CLAY1S [ 10 ] is a Modified Cam Clay (MCC) [ 11 ] type model that accounts for plastic anisotropy and destructuration. Anisotropy of plastic behavior is represented through an inclined and distorted elliptical yield surface and a rotational hardening law to model the development or erasure of fabric anisotropy during plastic straining, while interparticle bonding and degradation of bonds (structure) is reproduced using intrinsic and natural yield surfaces [ 12 ] and a hardening law describing destructuration as a function of plastic straining. The last letter of the model (“S”) refers to the soil structure. Thus, when the hierarchical version of the model without destructuration is used, the model is simply called S-CLAY1 [ 3 ]. For the sake of simplicity, the mathematical formulation is presented in the following in triaxial stress space, which can be used only to model the response of cross-anisotropic samples (cut vertically from the soil deposit) subject to oedometer or triaxial loading. The original inclined yield surface of the S-CLAY1 model is elliptical [ 3 ] (see Figure 1 ): f y = 1 + ( η − α ) 2 M 2 − α 2 − p m i ′ p ′ = 0 (9) where η is the stress ratio, M is the stress ratio at critical state, p ′ is the mean effective stress, p m i ′ is the size of the intrinsic yield surface related to the soil’s pre-consolidation pressure and α is the plastic anisotropy parameter that gives the inclination of the yield surface. In triaxial stress space, α is a scalar value, but, in the full three-dimensional formulation, α is a fabric tensor (3 × 3). The effect of bonding in the S-CLAY1S model [ 10 ] is described by an intrinsic yield surface [ 12 ] that has the same shape and inclination of the natural yield surface but with a smaller size (see Figure 1 ). The size of the intrinsic yield surface is specified by parameter p m i ′ which is related to the size p m ′ of the natural yield surface by parameter χ as the current amount of bonding p m ′ = ( 1 + χ ) p m i ′ (10) S-CLAY1S model incorporates three hardening laws. The first of these is the isotropic hardening law similar to that of MCC model [ 11 ] that controls the expansion or contraction of the intrinsic yield surface as a function of the increments of plastic volumetric strains ( d ε v p ) d p m i ′ = v p m i ′ λ i − κ d ε v p (11) where v is the specific volume, λ i is the gradient of the intrinsic normal compression line in the compression plane ( ln p ′ − v space), κ is the slope of the swelling line in the compression plane, and d ε v p is the increment of plastic volumetric strain. The second hardening law is the rotational hardening law, which describes the rotation of the yield surface with plastic straining [ 3 ] d α = ω [ ( 3 η 4 − α d ) ε v p ⟩ d d p (12) where d ε d p is the increment of plastic deviatoric strain, and ω and ω d are additional soil constants that control, respectively, the absolute rate of rotation of the yield surface toward its current target value, and the relative effectiveness of plastic deviatoric strains and plastic volumetric strains in rotating the yield surface. The third hardening law in S-CLAY1S model is for destructuration, which describes the degradation of bonding with plastic straining. The destructuration law is formulated in such a way that both plastic volumetric strains and plastic shear strains tend to decrease the value of the bonding parameter χ towards a target value of zero [ 10 ], it is defined as d χ = − ξ χ ( ε v p \| + ξ d \| d ε d p \| ) where ξ and ξ d are additional soil constants. Parameter ξ controls the absolute rate of destructuration, and parameter ξ d controls the relative effectiveness of plastic deviatoric strains and plastic volumetric strains in destroying the inter-particle bonding [ 13 ]. The elastic behavior in the model is formulated as isotropic, with elastic increments of volumetric and deviatoric strains given by the same expressions as in MCC: [ d ε v e d ε d e ] = [ 1 / K 0 0 1 / 3 G ] [ d p ′ d q ] (14) where K is a stress-dependent elastic bulk modulus ( K = ν p ′ / κ ). The model has traditionally been implemented assuming a constant Poisson’s ratio, v [ 3 , 14 ]. Thus, the shear modulus G is related to the elastic bulk modulus K , and is also stress dependent, instead of being constant. S-CLAY1S has proven its ability to reproduce the behavior of lightly overconsolidated structured soft clays both for laboratory tests and for boundary value problems [ 15 , 16 , 17 ]. The main advantages of the S-CLAY1S model are: (i) its relatively simple model formulation; (ii) its realistic K 0 prediction; and, most importantly; (iii) the fact that model parameter values can be determined from standard laboratory tests using well-defined methodologies [ 10 ]. S-CLAY1S was chosen to keep the model simple, but there are more advanced constitutive models for soft clays that, for example, introduce some flexibility in the shape of the plastic potential [ 14 ] or use a non-associated flow rule [ 18 , 19 ]. Some other advanced features of clay behavior, such as creep [ 20 ] or small-strains [ 21 ], have not been here considered either. 3. Stress-Dependent Elastic Cross-Anisotropy 3.1. Linear Elastic Cross-Anisotropy Fully generalized elastic anisotropy involves 21 independent elastic parameters due to the symmetry of the 6 × 6 stiffness matrix that relates the six independent stress increment components with the six independent strain increment components. The elastic matrix should be symmetric to satisfy thermodynamic principles [ 22 , 23 ]. However, most materials present some kind of symmetry that allows reducing the number of independent elastic parameters. Soil properties are influenced by their deposition and stress history, which are controlled by the gravity acceleration acting on the vertical axis (deposition and one-dimensional consolidation). Thus, soft clays that have not undergone any other types of straining, such as those caused by tectonic movements, present a cross-anisotropic or transversely isotropic behavior, i.e., both horizontal axes are interchangeable. That reduces the number of independent elastic parameters to 5, which are often chosen as E V , E H , ν VV , ν VH and G VH , where V and H subscripts refer to vertical and horizontal directions, respectively. The compliance matrix that relates the stress increments and the strain increments (inverse of the stiffness matrix) is as follow y d τ y z τ z (15) Rectangular Cartesian axes ( x , y , z ) are here used and z is taken as the vertical direction. While the vertical and horizontal stiffnesses ( E V ) have a clear physical meaning, distinguishing between different Poisson’s ratios and adding an independent shear modulus adds complexity to the study. Graham and Houlsby [ 6 ] showed that only three parameters can be deduced from triaxial tests on vertically cut specimens and proposed a simplified three parameter model ( E , ν , α e ). This is also called one-parameter anisotropy because only one parameter is added to describe anisotropy. Here, the subscript “ e ” is introduced in the α parameter to indicate that it refers to the elastic part and to distinguish it from the α parameter of the S-CLAY1S model that controls anisotropy in the plastic part. The stiffness matrix of this model is Comparing Equation (17) with Equation (15), it can be established that E , E H α e E * , ν H , , and G H V E . 3.2. Effects of Elastic Cross-Anisotropy in Triaxial Tests For triaxial tests, Graham and Houlsby [ 6 ] showed that the complete stiffness matrix (Equation (16)) reduces to: shows that there is some cross-coupling between volumetric and distortional effects, contrary to what happens under isotropic elasticity (Equation (14)). Manipulating the stiffness matrix (Equation (16)), , and may be expresses in terms of E , ν , and α e [ 24 ] (p. 49): The coupling between volumetric and distortional effects implies that: Under constant volume effective stress paths ( e ), the mean effective stress is no longer constant ( p 0 ), i.e., the stress path in the p’ : q plane is no longer vertical and depends on the value of α e [ 6 , 24 ] as follows (using Equations (18), (20) and (21)) q d p ′ = 3 G ). For example, for isotropic compression ( ), the ratio between shear and volumetric strains depends on the value of [ 6 , 24 ] as follows (using Equations (18), (20) and (21)) 3.3. Proposed Stress-Dependend Cross-Anisotropy 6 ] has been chosen as the basis for the current stress-dependent model because it allows to keep and with a similar meaning and the same values as the traditional and parameters. Besides, the only additional parameter (elastic anisotropic parameter, ) has a clear physical meaning ( ) and may be analytically obtained from conventional triaxial test, either from the undrained stress path during shearing (Equation (22)) or from the shear-volumetric strain ratio during drained isotropic compression (Equation (23)). Graham and Houlsby [ 6 ] model cannot be regarded as a full constitutive model and is only used for a very limited range of strains in soft soils because it does not consider stress-dependent stiffness nor plastic strains. The stress-dependent stiffness of the present model has been introduced relating the E* parameter with through a linear dependency on the effective mean pressure . To preserve the original meaning of as the slope of the swelling line in the compression plane ( space), the corresponding one-dimensional (confined) compression modulus has been used ( z , which can be obtained from Equation (16) taking ( + ν * 1 ν (24) For isotropic elasticity ( α e = 1 ), Equation (24) reduces to the bulk modulus d p ′ d ε z z e = E 3 ( 1 − 2 ν ) = K (25) The stress-dependent stiffness of a soil under unloading or reloading in the compression plane is given by e d p ′ = − κ p ′ (26) where the variation of the void ratio that corresponds to the elastic part ( d e e ) may be related to the increment of the vertical elastic strain d ε z z e d z e = − d e e 1 + e (27) Thus, using Equations (24), (26) and (27), the stress-dependent expression for the E* parameter is obtained E * = 3 ( 1 + p ′ (28) The stress-dependent elastic cross-anisotropy used in the present model is given by Equations (17) and (28). The present formulation of elastic anisotropy is hierarchical because if α e = 1 , the model reduces to S-CLAY1S [ 10 ] with isotropic elasticity. The current formulation of elasticity may be considered as hypoelastic [ 25 ], as it is defined in terms of stress and strain increments (Equation (17)) The authors are aware that this model is non-conservative [ 26 ], but assuming a constant Poisson’s ratio is widely used [ 3 ] and the differences for non-cyclic loads are small for practical purposes [ 27 ]. Application of a hyperelastic formulation [ 28 ] can solve the problem. However, most hyperelastic models [ 28 , 29 ] are fundamentally isotropic and only predict some stress-induced coupling between volumetric and deviatoric parts [ 30 ] (p. 151). A general stress formulation [ 30 ] (p. 153) may be used to model stress-induced anisotropy. Both stress-induced coupling [ 28 , 29 ] and stress-induced anisotropy predict an isotropic elastic response when the stress state is at the isotropic compression line. To properly predict strain-induced anisotropic elasticity within a hyperelastic formulation, a coupled elastic-plastic formulation is required, i.e., the energy potential should depend on the plastic strains [ 9 ], and that leads to a highly complex model. On the other hand, it is worth noting that results of oedometer tests are often plotted using a decimal logarithm scale (i.e., with base 10) for the vertical stress on the horizontal axis. For the virgin compression line, the relationship between the compression index C c and λ is exact ( C c ). However, for the unloading-reloading path, there is not an exact relationship between the swelling index and , because the ratio of horizontal and vertical stresses changes during one-dimensional unloading. If the ratio of horizontal and vertical stresses were the same as for the virgin compression line, the relationship would be . However, it is more realistic to assume that this ratio increases, and if an average value of 1 is adopted, i.e., horizontal stress approximately equal to vertical stress, the relationship is C s ≈ κ · ( l o g 10 e ) / 2 . 4. Validation The validation aims to shown the capabilities of the proposed model, which includes anisotropic elasticity in a simple way. The results predicted by the model are compared with laboratory tests on a well-documented soft natural clay, namely Bothkennar clay [ 31 , 32 , 33 ]. The results of the proposed model are compared with the results of S-CLAY1 ( α e = 1 ) to highlight the influence of anisotropic elasticity. Parameter calibration for S-CLAY1 is based on the method proposed by Wheeler et al. [ 3 ]. The additional parameter of the proposed model ( α e ) is estimated using Equations (22) or (23). To simplify the validation, soil destructuration has been initially neglected ( χ = 0 ), i.e., S-CLAY1 instead of S-CLAY1S. The Bothkennar soft clay test site is located on the south side of the Firth of Forth, in Scotland, and has been the subject of a number of comprehensive studies [ 31 , 32 , 33 ]. The Bothkennar clay is a dark grey/black micaceus clayey silt, in part thinly laminated and mottled, that was deposited in a stable marine environment. Typically, in a structured soil, the in situ water content is close to the liquid limit. The Bothkennar clay is remarkably uniform in composition and mineralogy but exhibits natural variability in structure, fabric and state. It has a sand content of less than 10% and a clay fraction generally of around 30% [ 32 ]. The principal clay mineral is illite, but the plasticity (plasticity index in the range 25–55%) appears enhanced by organic residues (3–8%), mainly from marine organisms. The mineral assemblage is predominantly quartz and feldspar flour, kaolinite and illite/mica [ 33 ]. The sensitivity varies with the facies type, being around 5–8 in the mottled facies and between 7 and 13 in the bedded and laminated facies [ 33 ]. Table 1 shows the adopted parameters for the proposed model. e 0 is the initial void ratio and σ ′ V c is the preconsolidation stress. The additional parameter of the proposed model ( α e = 1.3 ) was analytically obtained using Equation (23) from the ratio of deviatoric to volumetric strain increments ( d ε d e / d ε v e = 0.2 ) measured in the laboratory [ 31 ] during drained isotropic compression at pressures lower than the preconsolidation one, i.e., initial straight part in Figure 2 . α e = 1.3 means that the ratio of horizontal to vertical elastic stiffness is equal to E H / E V = . Those values are rounded to one decimal due to the lack of higher precision. Figure 2 shows how the proposed model reproduces the deviatoric strains during isotropic compression. Initially, during the elastic part, positive deviatoric strains ( ) are predicted because the higher horizontal elastic stiffness ( ). After yielding, the deviatoric strain increments are mainly plastic and negative ( ). Negative plastic deviatoric strain increments are predicted by the model because the yield surface is rotated and an associated plastic flow rule is adopted (see Figure 1 ). Obviously, yielding is sharply represented by the constitutive model, while the laboratory results [ 31 ] show a smooth transition. As plastic strains develop during isotropic loading, plastic anisotropy demises, the yield surface rotates toward the hydrostatic axis and plastic deviatoric strains tend to be 0 ( ). If elastic anisotropy is considered, deviatoric strains tend to be positive again as the laboratory results show for the final part ( Figure 2 ). This is clearer if an unloading-reloading loop is performed at the end of the test as done in test B7 by McGinty [ 31 ] ( Figure 3 ). The constitutive model with isotropic elasticity predicts a vertical straight line ( ) for the unloading-reloading loop, while an inclined straight line is predicted when anisotropic elasticity is included. The strain path direction is the same as that at the beginning of the test ( ) for the proposed constitutive model because a constant elastic anisotropy is adopted, while McGinty [ 31 ] data show a demise of elastic anisotropy ( ). An anomaly in McGinty [ 31 ] data is that no detectable change in strain path direction associated with the yield point is observed after reloading. This anomaly may be attributed to testing imperfections at high strains (>15%). For drained isotropic loading, the study has focused on the strain paths because the differences in the stress–strain behavior are small ( Figure 4 ). The results are slightly different because the elastic bulk modulus slightly depends on (Equations (19) and (28)). McGinty [ 31 ] performed drained triaxial tests at different stress ratios (see Table 2 ). The ratio of deviatoric to volumetric strain increments ( ) was approximately determined from both the initial elastic part and from the unloading stage for isotropic compression ( Table 3 ) and for different stress ratios ( Table 2 ). Although the determination for the initial elastic part is difficult as plastic strains quickly appear and it is difficult to ensure a true cut off point between pre-yield and post-yield behavior, Table 2 and Table 3 show a demise in the elastic anisotropy, i.e., for the unloading phase is roughly half of the initial value. The results are summarized in Figure 5 . In the proposed model, the ratio depends on the stress ratio ( ) as follows: (29) For the isotropic case ( ), the ratio linearly varies with the stress ratio ( ): (30) Atkinson et al. [ 34 ] present results of undrained triaxial compression tests carried out on samples of intact Bothkennar clay recovered using a Laval sampler. The laboratory test samples were cut from the Laval sample using different techniques. As shown in Figure 6 , the effective stress path in the p’ : q plane is no longer vertical for the initial overconsolidated (elastic) part due to the anisotropic elastic behavior. As previously mentioned, under constant volume effective stress paths ( ε v e = 0 ), the mean effective stress is no longer constant ( d p ′ 0 ) and depends on the value of α e (Equation (22)) for the proposed anisotropic model. The fitted value α e = 1.3 analytically obtained using Equation (23) and McGinty [ 31 ] data agrees with that obtained using Equation (22) and Atkinson et al. [ 34 ] data. The depth of the samples used by Atkinson et al. [ 30 ], namely 12.6 m, is slightly higher than those samples used by McGinty [ 31 ] (around 11 m depth). Thus, the parameters used for the simulations shown in Figure 6 a are those in Table 1 , but for the presconsolidation stress ( V = 105 kPa). To improve the agreement between laboratory results and the proposed model, numerical simulations including destructuration (S-CLAY1S) are also depicted in Figure 6 b. The parameters used to model destructuration are shown in Table 4 . The initial elastic part is not affected when destructuration is included, but the final plastic part shows a notable reduction in the stress level along the critical state line in agreement with the laboratory tests. Here validation limits to Bothkennar clay, but similar results would be obtained for other soft clays, for example, Winnipeg clay [ 6 ]. Winnipeg clay is a post-glacial plastic clay from Lake Agassiz, Canada. The clay-size fraction is often as much as 75–80%, while the clay minerals are mostly interlayered montmorillonite-illites [ 35 ]. Although both Bothkennar and Winnipeg clay are soft natural structured clays, most of their properties are different, e.g., the drained strength and plastic anisotropy [ 3 ]. The anisotropic elastic parameter slightly varies with the depth sample for Winnipeg clay andits value is around 1.2–1.4 [ 6 ]; thus, it is similar to that obtained for Bothkennar clay (1.3). 5. Parametric Analyses To highlight the influence of some of the model parameters, particularly the anisotropic elastic parameter , parametric analyses were performed. Figure 7 shows the increments of the effective stress paths for undrained triaxial compression tests. The results and parameters in Figure 7 are based on the undrained triaxial tests performed by [ 34 ] and presented in Figure 6 . Since the focus is on the influence of the anisotropic elastic parameter on the initial slope of the effective stress path, the increments of deviatoric and mean stresses are presented in Figure 7 . The slope of the effective stress ratio has limiting values of −1.5 and +3 for very large and very small values of , respectively. The variation of the slope of the effective stress path with the anisotropic elastic parameter is given by Equation (22) and plotted in Figure 8 for several values of the Poisson’s ratio. The relationship is valid for the presented model and for the linear elastic model by Graham and Houlsby [ 6 ]. On the other hand, the slope of the strain paths (ratio between deviatoric and volumetric strains) for isotropic compression is given by Equation (23) for the presented model and for the linear elastic model by [ 6 ]. The relationship is plotted in Figure 9 . The slope of the strain path has limiting values of 2/3 and −1/3 for very large and very small values of α e , respectively. Using the case presented in Figure 2 as a basis, a parametric analysis is presented in Figure 10 . The anisotropic elastic parameter ( α e ) is slightly varied between 1.2 and 1.4. It is remarkable that α e influences the slope of the strain path not only for the initial elastic part, but also for the final plastic part, where the elastic strains have a non-negligible influence on the slope of the strain paths. Figure 10 also shows the influence of the parameter ω that controls the absolute rate of rotation of the yield surface toward its current target value (Equation (12)). For isotropic loading, the target value is α = 0 , i.e., a non-inclined yield surface and null plastic deviatoric strains because an associated flow rule is used in the proposed model. Thus, for ω = 50, the yield surface rotates faster to the horizontal (hydrostatic) axis ( Figure 1 ), deviatoric plastic strains are quickly 0 and the strain path quickly bends to the right again due to the positive deviatoric elastic strains. However, the slopes of the initial (elastic) part and the final (plastic) part are not the same because of the different values of the elastic and plastic volumetric strains. 6. Conclusions A novel constitutive model for soft structured clays that includes anisotropic behavior both of elastic and plastic nature has been developed. The new model incorporates stress-dependent cross-anisotropic elastic behavior within the yield surface using only an additional parameter to describe anisotropic elasticity, namely the square root of the ratio between horizontal and vertical stiffnesses. Comparisons with laboratory results for Bothkennar clay show the procedure to analytically obtain the additional anisotropic elastic parameter from conventional triaxial tests and the improved capabilities of the presented model, namely the initial non-vertical effective stress paths for undrained triaxial tests and the non-null deviatoric strains during isotropic loading or unloading. The proposed model clearly improves the predictive capabilities of its parent constitutive model (S-CLAY1S) at an element test level without notable complexity. Further validation of the model should be performed for boundary value problems. Acknowledgments The authors thank S. Wheeler and M. Karstunen for their advice and for providing raw data of triaxial tests on Bothkennar clay. The authors of this work would like to express their gratitude to the Spanish Ministry of Economy, Industry and Competitiveness for the financial support of the project BIA2015-67479-R: “The critical distance in rock fracture”, on the results of which this paper is based. This research project is supported by the European Regional Development Fund of the European Union. Author Contributions J.C. contributed to the conception and further development of the model, its validation and parametric analyses as well as writing the paper. 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Yield surfaces of the S-CLAY1S model (Modified from [ 10 ]). Figure 2. Influence of elastic anisotropy during isotropic compression (thin blue lines are laboratory results [ 31 ] and thicker red and black lines are S-CLAY1 predictions using isotropic and anisotropic elasticity, respectively). Figure 2. Influence of elastic anisotropy during isotropic compression (thin blue lines are laboratory results [ 31 ] and thicker red and black lines are S-CLAY1 predictions using isotropic and anisotropic elasticity, respectively). Figure 3. Influence of elastic anisotropy for an isotropic unloading-reloading loop. Figure 3. Influence of elastic anisotropy for an isotropic unloading-reloading loop. Figure 4. Stress–strain behavior during isotropic loading. Figure 4. Stress–strain behavior during isotropic loading. Figure 5. Variation of strain path direction with stress ratio (Ini.: initial loading; Unload.: unloading). Figure 5. Variation of strain path direction with stress ratio (Ini.: initial loading; Unload.: unloading). Figure 6. Effective stress path for undrained triaxial compression: ( a ) simulations using S-CLAY1; and ( b ) simulations using S-CLAY1S (considering destructuration). Figure 6. Effective stress path for undrained triaxial compression: ( a ) simulations using S-CLAY1; and ( b ) simulations using S-CLAY1S (considering destructuration). Figure 7. Effective stress path for undrained triaxial compression (each line corresponds to the anisotropic elastic parameter, α e , that is indicated over the line). Figure 7. Effective stress path for undrained triaxial compression (each line corresponds to the anisotropic elastic parameter, α e , that is indicated over the line). Figure 8. Elastic effective stress path for compression under constant volume ( Figure 8. Elastic effective stress path for compression under constant volume ( Figure 9. Strain path (ratio between shear and volumetric strains) for compression under constant shear stresses ( Figure 9. Strain path (ratio between shear and volumetric strains) for compression under constant shear stresses ( Figure 10. Strain path (ratio between shear and volumetric strains) for compression under constant shear stresses ( Figure 10. Table 1. Model parameters for Bothkennar clay. Table 1. Model parameters for Bothkennar clay. 1 For a reference isotropic pressure of 15 kPa; 2 Using Equation (23) and triaxial data after McGinty [ 31 ]. Table 2. Strain path directions for triaxial tests at different strain ratios [ 31 ]. Table 2. Strain path directions for triaxial tests at different strain ratios [ 31 ]. Test C1 C2 C3 C4 C5 C6 C7 C9 η 0.42 1.11 1.30 −0.80 0.80 0.20 0.42 −0.50 d ε d e / d ε v e 1 0.36 0.75 0.92 −0.65 0.53 0.2 0.53 −0.28 d ε d e / d ε v e 2 0.25 0.38 0.47 −0.31 0.25 0.16 0.23 −0.06 Table 3. Strain path directions for isotropic loading [ 31 ]. Table 3. Strain path directions for isotropic loading [ 31 ]. Test B1 B2 B3 B4 B5 B6 B7 B8 η 0 0 0 0 0 0 0 0 d ε d e / d ε v e 1 0.20 0.20 0.20 0.20 0.14 0.18 0.21 0.31 d ε d e / d ε v e 2 0.08 0.14 0.18 0.20 0.15 0.10 0.12 0.07 1 Initial loading; 2 Unloading. Table 4. Additional model parameters to include destructuration for Bothkennar clay. Table 4. Additional model parameters to include destructuration for Bothkennar clay. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/ ). AMA Style Castro J, Sivasithamparam N. A Constitutive Model for Soft Clays Incorporating Elastic and Plastic Cross-Anisotropy. Materials. 2017; 10(6):584. https://doi.org/10.3390/ma10060584 Materials , EISSN 1996-1944, Published by MDPI	https://www.mdpi.com/1996-1944/10/6/584
Behind the Label: Inequality in the Los Angeles Apparel Industry \| Request PDF Request PDF \| On Jan 1, 2000, Edna Bonacich and others published Behind the Label: Inequality in the Los Angeles Apparel Industry \| Find, read and cite all the research you need on ResearchGate Article Behind the Label: Inequality in the Los Angeles Apparel Industry Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations ... These coalitions brought together a mosaic of community groups acting for the defense of human rights, women, consumers, immigrants, workers, as well as religious groups, student groups, teacher associations, and in some cases, lawyers and political organizations. The rise of such 'anti-sweatshop movement' in the clothing sector has been well documented with reference to the US (Bonacich and Appelbaum 2000; Bender and Greenwald 2003;Esbenshade 2004), and is part of a broader 'globalization from below' by which counter-hegemonic groups have developed forms of 'transnationally organized resistance' against the unequal exchanges produced or intensified by economic globalization (Sousa Santo and Rodriguez-Garavito 2005;Sousa Santos 2006). In characterizing the varied forms and dynamics that such organized resistance might take, Della Porta and Tarrow (2005) insist on the long term, intense interactions observed in transnational collective actions defined as 'coordinated international campaigns on the part of networks of activists against international actors, other states, or international institutions" (p. 7). ... ... Other similarities between contemporary campaigns and the social dynamics observed in the formative stages of the Fordist period can be seen in the adoption of a confrontational attitude vis-à-vis large corporations, aimed at promoting a Selznickian 'legitimate selfassertion' of production workers in GCCs (Bonacich and Appelbaum 2000; Sum and Pun Ngai 2005). As a consequence, classic dilemmas historically faced by labor unions in terms of arbitration between confrontational and cooperative attitudes vis-à-vis corporations have arisen in global counter-power networks as well (Utting 2005a). ... ... they stopped doing so during the 1980 and 1990s by adopting individualized compensation schemes and/or subcontracting work to employers and places where Fordist counter-powers, i.e., the State and labor union, were weakly organized or inexistent (Bonacich and Appelbaum 2000; Capelli 1999). While economic decisions became increasingly centralized in global management systems, societies remained organized -and redistributive institutions continued to operate -on a national basis. ... Challenging Governance in Global Commodity Chains: The Case of Transnational Activist Campaigns for Better Work Conditions Chapter Full-text available Jan 2010 Florence Palpacuer The coordination or ‘functional integration’ of complementary activities across locations constitutes a distinctive feature of globalization in contemporary capitalism (Dicken 1998). In industries such as apparel, electronics and automobiles, it has led to the emergence of ‘Global Commodity Chains’ (GCCs) governed by large lead firms that retain direct control over marketing and design activities in Northern markets while arranging for the manufacture of their products in complex transnational networks spanning Southern countries. Although mainstream economists continue to think of globalization in terms of international competition, seeing the surge of manufacturing imports in mature markets as resulting from the superior cost advantage of Southern producers, a GCC perspective emphasizes that the globalization of production has been driven primarily by large firms in the North taking advantage of information and communication technologies, transport deregulation, trade liberalization, and an abundant labour supply in the South to reorganize production across countries and world macroregions in order to lower cost, increase flexibility, and build up scale (Gereffi 1994). ... The global value chain/production network literature assumes some degree of power asymmetry in supply chains (Dicken, 2015;Gereffi, Korzeniewicz, & Korzeniewicz, 1994). This imbalance is perhaps most noticeable in buyer-driven apparel global supply chains (GSCs), where retailers and brands ('buyers' or 'lead firms') consolidated their power through mergers, acquisitions and market concentration (Abernathy, Dunlop, Hammond, & Weil, 1999; Bonacich & Appelbaum, 2000) . At the same time, there has been a dramatic dispersion of apparel suppliers across developing countries (Gereffi & Frederick, 2010). ... ... One of the original premises of the GSC 1 literature is that there are power imbalances across different nodes of chains Henderson, Dicken, Hess, Coe, & Yeung, 2002). Bonacich and Appelbaum (2000) find that high levels of competitiveness among supplier factories and mergers among retailers increased the power of retailers over suppliers. Abernathy et al. (1999) come to similar conclusions about the trend toward retailer consolidation, but for different reasons. ... Squeezing workers’ rights in global supply chains: purchasing practices in the Bangladesh garment export sector in comparative perspective Article Jun 2019 Mark S. Anner Workers’ rights violations have been pervasive in many global supply chains. In the apparel sector, production workers often face precarious working conditions, including persistently low pay, excessive and often forced overtime, unsafe buildings, and repression of their right to form unions and bargain collectively. This article explores how purchasing practices of lead firms adversely affect working conditions and workers’ rights in supplier factories. It attributes these trends to a price squeeze and a sourcing squeeze in which lead firms pay increasing lower prices to suppliers while also imposing short lead times and high order volatility. To test this argument, trade data of apparel imports to the United States and the European Union are explored. The article then turns to original surveys of Bangladesh supplier factories and workers carried out in 2016 and 2017. The final section of this paper examines the impact of the squeeze on working conditions and workers’ rights using the Labour Rights Indicators. ... Há já vários estudos que enfatizam a relação entre a migração internacional e o trabalho no setor de confecção nas grandes cidades dos Estados Unidos, Argentina e Brasil (Benencia, 2009; Bonacich; Appelbaum, 2000;9 Confederação Nacional dos Trabalhadores nas Indústrias do Setor Têxtil, Vestuário, Couro e Calçados -Entrevista realizada em 12/05/2015. Montero, 2008;Sassen, 1989;Truzzi, 2008), os quais evidenciam o tipo de trabalho precário desenvolvido no que se convencionou chamar de sweat shops. ... ... Há já vários estudos que enfatizam a relação entre a migração internacional e o trabalho no setor de confecção nas grandes cidades dos Estados Unidos, Argentina e Brasil (Benencia, 2009;Bonacich; Appelbaum, 2000; 9 Confederação Nacional dos Trabalhadores nas Indústrias do Setor Têxtil, Vestuário, Couro e Calçados -Entrevista realizada em 12/05/2015. Montero, 2008;Sassen, 1989;Truzzi, 2008), os quais evidenciam o tipo de trabalho precário desenvolvido no que se convencionou chamar de sweat shops. ... O TRABALHO NA CONFECÇÃO EM SÃO PAULO: as novas formas da precariedade1 Article Full-text available Apr 2017 Marcia Leite Sandra Roberta Alves Silva Pilar Carvalho Guimarães This article analysis the work in the clothing sector based on a research made in the city of São Paulo. This article is focused specifically on home jobs and on the work of immigrants. It is discussed the working conditions in the sector from the configuration of value chains and from the international division of work that it promotes generating a new dynamics in the forms of precariousness that have always marked the sector. This study also discusses the impact of the Brazilian Textile Retail Association (ABVTEX) on the conditions of sewing workers at their homes and at factories ... It was facilitated in part by the Hart-Celler Act of 1965, which struck from the books the last vestiges of the national originbased quotas that had barred most immigration from Asia and instituted 20 Even prior to the United Kingdom's departure from the European Union (EU), the size of the EU's unauthorized population was less than half of that of the United States, despite the EU's overall population being nearly two thirds greater (Connor et al., 2019;Passel & Cohn, 2018). 21 For an excellent study of these processes from the perspective of one industry heavily reliant on undocumented labor, see Bonacich and Appelbaum (2000) . family reunification as a cornerstone principle of US immigration policy. ... Marxism and Migration Book Nov 2022 Sara Carpenter Genevieve B Ritchie Shahrzad Mojab ... It was facilitated in part by the Hart-Celler Act of 1965, which struck from the books the last vestiges of the national originbased quotas that had barred most immigration from Asia and instituted 20 Even prior to the United Kingdom's departure from the European Union (EU), the size of the EU's unauthorized population was less than half of that of the United States, despite the EU's overall population being nearly two thirds greater (Connor et al., 2019;Passel & Cohn, 2018). 21 For an excellent study of these processes from the perspective of one industry heavily reliant on undocumented labor, see Bonacich and Appelbaum (2000) . family reunification as a cornerstone principle of US immigration policy. ... As Migrants Move: (Re)formation of Class and Class Struggle Chapter Jan 2022 Genevieve B Ritchie Sara Carpenter Shahrzad Mojab This chapter provides a brief overview of the research that inspired Marxism and Migration. The analysis situates the collection within various Marxian traditions and lays out the material conditions that coalesce into displacement, movement, and mobility. The chapter provides both a rationale for the collection and a foundation for thinking through the realities of struggle. ... It was facilitated in part by the Hart-Celler Act of 1965, which struck from the books the last vestiges of the national originbased quotas that had barred most immigration from Asia and instituted 20 Even prior to the United Kingdom's departure from the European Union (EU), the size of the EU's unauthorized population was less than half of that of the United States, despite the EU's overall population being nearly two thirds greater (Connor et al., 2019;Passel & Cohn, 2018). 21 For an excellent study of these processes from the perspective of one industry heavily reliant on undocumented labor, see Bonacich and Appelbaum (2000) . family reunification as a cornerstone principle of US immigration policy. ... Marxist Perspectives on Migration Between Autonomy and Hegemony: An Intervention for a Strategic Approach Chapter Jan 2022 Leandros Fischer Martin Bak Jørgensen This contribution represents a critical evaluation of Marxist perspectives on migration. It focuses especially on the so-called “autonomy of migration” approach, probably the most influential Marxist-inspired theory in the field of critical migration studies. Tracing its origins in Italian and German traditions of operaismo, we particularly take issue with its assumption of decentred world order, as well as its overt focus on the subjective capacity of migrants. Proposing a more complex perspective of structure and agency, we propose a Gramscian perspective on migration that considers the role of civil society alliances between migrants and those in solidarity with them. ... disenfranchised persons who are hence highly vulnerable (Bender & Greenwald, 2003; Bonacich & Appelbaum, 2002; Ross, 2004). I will first dwell on the implications of the Chinese household registration system to illustrate the extent to which Chinese workers are being marginalised to achieve economic growth (Section 3), and how this system increases political and economic control (Section 3.1.). ... Politics of Sweatshop Labour Article Full-text available Apr 2022 Janelle Pötzsch This paper provides a political account of sweatshop labour by highlighting the role of political actors in establishing and sustaining exploitative working conditions in global sweatshops. In academic debates on sweatshop labor, the focus has been mostly on the responsibility of business or consumers (cf. Arnold and Bowie 2007, Meyers 2007, Zoller 2015), albeit there are some authors who deal with the role of trade unions and civil society (cf. Kabeer 2000). To provide a broader picture, I draw on the differentiation between extractive and inclusive institutions made by development economists Acemoglu and Robinson (2013) and apply this account to the sweatshop issue. I will argue that sweatshops are a political rather than an economic issue, and substantiate my claim by drawing on the Chinese household registration system hukou, and the political background conditions in Bangladesh. ... Governments made decisions to liberalize financial markets (Foroohar, 2017), phase out trade restrictions in the apparel sector (Gereffi & Frederick, 2010), and promote EPZs through development assistance and tax incentives (Anner, 2011). Moreover, the use of technology by industry leaders (notably, the use of big data to determine product demand and to facilitate speed to market), further consolidated the power of lead firms (Abernathy et al., 1999; Bonacich & Appelbaum, 2000; Lichtenstein, 2009 ... The Governance Challenges of Social Upgrading in Apparel Global Value Chains in the Context of a Sourcing Squeeze and the Covid-19 Pandemic Chapter Jan 2022 Mark S. Anner Freedom of association and living wages are two fundamental components of social upgrading in global value chains. However, in the apparel sector, the power afforded to lead firms due to industry consolidation has resulted in a squeeze on suppliers and on workers that is reflected in terms of low wages and increased worker rights violations. This squeeze down the supply chain has been exacerbated by the Covid-19 pandemic, with many lead firms abruptly cancelling orders without paying supplier factories for their orders. Millions of workers were adversely affected through lost income, lost jobs, and increased food insecurity. The sourcing squeeze and the Covid-19 crisis thus illustrate decent work governance gaps in global value chains. This chapter argues that these gaps can be addressed via a transformation of the institutional context in which GVCs operate. This involves collective bargaining and other binding agreements and more effective public governance. These arguments are illustrated through the country case study of India. ... Governments made decisions to liberalize financial markets (Foroohar, 2017), phase out trade restrictions in the apparel sector , and promote EPZs through development assistance and tax incentives (Anner, 2011). Moreover, the use of technology by industry leaders (notably, the use of big data to determine product demand and to facilitate speed to market), further consolidated the power of lead firms (Abernathy et al., 1999; Bonacich & Appelbaum, 2000; Lichtenstein, 2009 ... Economic and Social Upgrading in Global Value Chains Comparative Analyses, Macroeconomic Effects, the Role of Institutions and Strategies for the Global South: Comparative Analyses, Macroeconomic Effects, the Role of Institutions and Strategies for the Global South Book Full-text available Jan 2022 Christina Teipen Petra Dünhaupt Hansjörg Herr Fabian Mehl “Finally, a set of analyses that explicitly brings power relations into the study of Global Value Chains. This excellent book highlights various dimensions of a critical but understudied feature: power asymmetries of both demand and supply at each stage, which give rise to rent seeking in the upper levels and reduce capacities for upgrading at the lower levels of the chain. Public policies are crucial in determining the outcomes, which makes this book essential reading for anyone interested in economic development.” — Jayati Ghosh, Professor of Economics, University of Massachusetts at Amherst, USA. “This book is an important contribution to the debate on global value chains. It is particularly important because it takes the perspective of the Global South and shows the risks for developing countries integrating through global value chains in a global economy with massive power imbalances. It also gives some hope that risks can be turned into success with strategic industrial and trade policies, social upgrading, strong international human rights due diligence regulations and a strong voice for workers.” — Atle Høie, General Secretary of IndustriALL Global Union This book investigates how global value chain governance, public institutions and strategies in the area of industrial policy and industrial relations by stakeholders such as national or global trade unions, governments, companies or international NGOs shape upgrading in the Global South. A special feature is its interdisciplinarity, combining sociological, economic, legal and political dimensions. Case studies systematically compare different industry trajectories. Furthermore, it encompasses far-reaching insights into the role of global value chains for development, economic catching-up of countries and socio-political aspects such as working conditions and interest representation. Christina Teipen is Professor for social sciences with a focus on economic sociology at HWR Berlin (Berlin School of Economics and Law). Petra Dünhaupt is a research associate and lecturer at HWR Berlin. Hansjörg Herr is Professor (retired) for supranational integration at HWR Berlin. Fabian Mehl is a research associate and lecturer at HWR Berlin. ... Governments made decisions to liberalize financial markets (Foroohar, 2017), phase out trade restrictions in the apparel sector , and promote EPZs through development assistance and tax incentives (Anner, 2011). Moreover, the use of technology by industry leaders (notably, the use of big data to determine product demand and to facilitate speed to market), further consolidated the power of lead firms (Abernathy et al., 1999; Bonacich & Appelbaum, 2000; Lichtenstein, 2009 ... Economic and Social Upgrading in Global Value Chains: The Automotive Industry in Brazil Chapter Jan 2022 Anselmo L. dos Santos José Dari Krein Denis MARACCI Gimenez Hugo Dias For over 20 years, the Brazilian economy has faced enormous challenges as a result of neoliberal structural reforms, economic stagnation and external crises, particularly after the Latin American crisis of the late 1990s to early 2000s and after 2014. Deep economic structural changes and fluctuations in the pace of growth profoundly affected Brazilian production and consumption, various sectors of activity, and the social and working conditions of the population. One of the largest and most historically significant industrial sectors in Brazil is the automotive industry. While working conditions in the industry have greatly improved since the beginning of the 2000s, with worsening macroeconomic conditions since 2014 the Brazilian labour movement’s ability to continue achieving an upwards trajectory for workers seems to be under threat. This analysis focuses on the automotive industry as a point of reference to better understand Brazil’s economic development since the late 1990s, the challenges inherent to integration in global value chains (GVCs) and how all of these factors influence working conditions and the ability of organised labour to achieve improvements. ... Costs are largely reduced by taking advantage of the cheap labor force in developing countries. Garment production is relatively labor intensive, requiring little start-up capital and can therefore be very easily delocalized in countries where labor cost is low (Bonacich & Appelbaum, 2000) . In 2004, developing countries accounted for nearly 75% of all clothing exports; the percentage was similar in 2017 (Bruce & Daly, 2006;World Trade Organization, 2018) In particular, H&M is the largest exporter of clothing from Bangladesh, and the Rana Plaza incident 4 highlighted the connections between mass fashion companies and the poor working conditions in clothing production companies in low-wage countries. ... Front-Line Workers in the Global Service Economy: Overshadowed and Overstretched in the Fast Fashion World Book Full-text available May 2021 Giovanna Fullin ... Respecto al rol de la inmigración, Bonacich et al. (2000) concluyen que el sistema de sweatshops es posible en Estados Unidos gracias a la presencia de inmigrantes latinos y asiáticos, muchos de ellos indocumentados, sin otras posibilidades de empleo producto de la segmentación racial que caracteriza dicho mercado laboral. La discriminación que sufren la mayoría de los inmigrantes restringe sus oportunidades laborales e incrementa su vulnerabilidad social, debilitando su poder de negociación para defender sus condiciones laborales, fenómeno también detectado en estudios para Argentina (por ejemplo, Lieutier, 2010). ... INFORMALIDAD LABORAL EN LA FABRICACIÓN DE ROPA EN ARGENTINA ENTRE 2003-2018:¿QUÉ APORTA LA ENCUESTA PERMANENTE DE HOGARES? LABOUR INFORMALITY IN THE MANUFACTURE OF CLOTHING IN ARGENTINA BETWEEN 2003-2018: WHAT DOES THE PERMANENT HOUSEHOLD SURVEY CONTRIBUTE? Article Full-text available Jul 2020 Estud. econ. Gustavo Ludmer Federico Favata A nivel mundial, la fabricación de ropa se caracteriza por las adversas condiciones laborales que sufren sus trabajadores. Con un abordaje metodológico cuantitativo, este artículo analiza la relación entre los atributos observables tanto individuales como del puesto de trabajo y la informalidad laboral del sector en Argentina entre 2003 y 2018. Utilizando los microdatos de la Encuesta Permanente de Hogares, se realizó una descomposición Oaxaca-Blinder para conocer los determinantes obser-vables de la problemática. Los resultados obtenidos sugieren que: i) los trabajadores del sector tienen mayor probabilidad de ser informales que el resto de los ocupados en Argentina; ii) dicha diferencia se explica principalmente por la mayor presencia relativa de trabajadores y puestos de trabajo con atributos usualmente penalizados en el mercado laboral; y iii) por una penalidad propia del sector, que aumenta las probabilidades de ser informal, aun controlando por dichos atributos. Código JEL: J24, J46, J81. Palabras clave: costureros, informalidad laboral, Oaxaca-Blinder. ... Como "la más global de todas las industrias" (Bonacich y Appelbaum, 2000) , la industria de la indumentaria es un claro ejemplo de estos procesos. Las firmas líderes han logrado reducir sus costos laborales mediante la deslocalización, permitiendo una aguda deflación en los precios que, acompañada de grandes inversiones en marketing y en localización de los comercios, ha llevado a un fabuloso incremento del consumo. ... Impacto de las importaciones de indumentaria en la producción y el empleo en Argentina (1990-2015) ¿Desindustrialización o informalización laboral? Article Full-text available Dec 2018 Jerónimo Montero Bressán Este artículo se propone analizar los efectos de la creciente importación de indumentaria en la producción y el empleo en la Argentina entre 1990 y 2015. Aquí se postula que la idea de que más importaciones implican destrucción de empleos es un tanto simplista por dos razones. En primer lugar, la relación entre ambas variables es compleja y requiere del análisis conjunto de otras variables, especialmente del consumo interno. En segundo lugar, el efecto de las importaciones no ha sido solo cuantitativo (menos empleo) sino también –y en mayor medida– cualitativo (empleo de menor calidad). Ante el explosivo aumento de las importaciones desde 1990, los empresarios locales cerraron las fábricas para dedicarse a generar un mercado de mayor valor agregado (ropa de moda), basado en la subcontratación de talleres clandestinos de costura o ‘maquilas locales’ que emplean a migrantes en condiciones de trabajo forzoso. Abstract This article examines the effects of the continuous growth of clothing imports on production and employment in Argentina from 1990 to 2015. Here I contest the idea that more imports mean employment destruction. On the one hand, the relationship between these variables is complex and requires the joint analysis of other variables –most notably the dynamics of consumption. On the other hand, the effect of garment imports has not only been quantitative (less employment), but it has also –and rather– been qualitative (employment in worse conditions). In facing bursting imports since 1990, local employers shifted to fashionwear production, shutting down their factories and subcontracting to ‘local sweatshops’ where migrants are subjected to practices of forced labour. ... Wal-Mart, which is less subject to consumer pressure than fashion brands such as Nike, overtly suppresses unions and drives down prices to the point where the factory social responsibility inspections are largely superficial, and according to the Institute of Contemporary Observation in China, "[n]inety percent of Wal-Mart sub-contractors and suppliers cannot meet Wal-Mart's own code of conduct" (Appelbaum & Lichtenstein, 2006, p. 11). Large retailers are at the top of the "pyramid of power" (Bonacich & Appelbaum, 2000) and can set the terms of contracts, squeezing every link in the chain down to the bottom with disregard for labor conditions. ... Compromised Stability And Security In The “Race To The Bottom” Article Christopher Wegemer Kellie Hinze ... Redclift and Sage (1998) pointed to increasing evidence that transnational organisations have a negative impact on the environment. Similarly, Ross (2004) blames the internationalisation of MNCs for the re-emergence of sweatshops in China, which is clearly encouraging a race to the bottom among emerging countries, and even developed countries (Bonacich and Appelbaum, 2000) . ... Racing to the bottom, or climbing to the top? Local responses to the internationalisation of trade in the Brazilian textile and garments industry Article Robson sø Rocha This article explores how firms in the textile and garment industry in Brazil are responding to trade liberalisation and the entry of multinational retailers into the Brazilian market. We analyse the competitive responses of local garment firms and, relatedly, how these responses are impacting on the industry's use of water. We show that the effects of trade liberalisation on the strategies of local firms and the use of natural resources are dependent upon the prior capacity of local actors to mobilise financial and human resources. In our case, contrary to the 'pollution havens' hypothesis, there is no indication that the regulatory framework has been downgraded to attract multinational companies (MNCs) or to help local firms cope with international competition. We claim that races to both the top and bottom are taking place as responses to the new competitive conditions. ... This imbalance is perhaps most noticeable in buyer-driven apparel global supply chains. This is because on the one end, retailers and brands ('buyers') have increasingly consolidated their power through mergers, acquisitions and market consolidation (Abernathy et al. 1999; Bonacich and Appelbaum 2000) . At the same time, there has been a dramatic dispersion of suppliers across developing countries. ... CSR Participation Committees, Wildcat Strikes and the Sourcing Squeeze in Global Supply Chains: CSR Participation Committees, Wildcat Strikes Article Mark S. Anner Lead firms in apparel global supply chains are increasingly using social compliance programmes that require worker-management participation committees in their supplier factories. These committees are designed to ensure respect for internationally recognized labour standards, to empower workers, and to reduce labour unrest. However, these committees have remained weak, and in countries such as Vietnam worker unrest remains common. This article argues that this is because lead firms in these CSR programmes are imposing a ‘sourcing squeeze’ on supplier factors by reducing the prices and production times they allot to their suppliers, which undermines efforts by committees to address cost-sensitive issues and overtime violations. At the same time, the sourcing squeeze increases strike leverage, providing workers with a much more effective source of worker voice. ... En este giro, dos cuestiones resultan de particular importancia. En primer lugar, el brusco cambio en el balance de poder entre capital y trabajo [Bonacich y Appelbaum, 2000; Harvey, 2007Harvey, (2005Moody, 1997]. En segundo lugar, una creciente neoliberalización de los Estados nacionales, que ha llevado a un giro en las capacidades y prioridades selectivas del estado[Jessop, 2008[Jessop, (2002. ... La moda neoliberal: El retorno de los talleres clandestinos de costura. En Geograficando, Vol 8 Article Full-text available Jerónimo Montero Bressán En este artículo se analiza el resurgimiento de talleres de costura clandestinos en grandes ciudades del centro y la periferia mundial, para entender los cambios dados en la industria de la moda durante las últimas cuatro décadas y sus consecuencias sobre los trabajadores. Para ello se realizaron dos estudios de caso: uno en la ciudad de Buenos Aires y otro en la provincia de Prato (Italia). Los resultados de esta investigación demuestran que este sector fue pionero en los procesos de reorganización industrial en la época neoliberal. En ambos estudios de caso, el cierre de fábricas y la utilización masiva de subcontratación a talleres urbanos informales tuvieron como consecuencias una significativa concentración de capital por un lado, y un marcado deterioro de las condiciones de trabajo por el otro. De hecho, el empleo de trabajadores migrantes víctimas de trata de personas y trabajo forzoso es fundamental para el funcionamiento de esta industria. This paper explores the re-emergence of sewing sweatshops both in core and peripheral big cities around the world, in order to understand the changes that took place in the fashion industry over the past four decades and their consequences on the workers. Two case studies were carried out to this aim: one in the city of Buenos Aires and another one in the province of Prato (Italy). The results show that changes in this industry illustrate industrial re-organisation processes in Neoliberal times. In both case studies, the closing down of factories and the subcontracting of informal urban workshop resulted in a deterioration of working conditions. In fact, the employment of thousands of migrant workers subjected to human trafficking and forced labour is vital to the fashion industry. View ... While the informalization of the production chain in the garment industry and the associated deterioration of labor conditions have been the subject of much research, studies on how these social constellations are locally fostered are few and far between. 4 The revival of sweatshops in many Asian countries, including Bangladesh, Indonesia, Sri Lanka, Cambodia, China, and Thailand, but also in Brazil and the United States is usually seen as part of a much broader phenomenon, namely, the restructuring of global capitalism (Esbenshade 2004;Sluiter 2009; Bonacich 2000; Adams 2002: 149). In the case of Argentina, such a reconfiguration of the global economic order refers to a set of processes, the final outcome of which has been the spread of informal labor relationships, as has been reflected in a clear decline of the manufacturing industrial complex since the mid-1970s (Kosacoff et al. 2004;Campos 2008terms, there are two key developments mediating the global tendencies identified as promoting informalization: one is the increasing income inequality that fosters dichotomous high-and low-income consumption patterns; the other is a growing inequality in profit-making among providers (Sassen 1994: 2290). ... Taxing the Shadow: The Political Economy of Sweatshops in La Salada, Argentina Working Paper Full-text available Nov 2014 Matías Dewey ... trueque en la actualidad, es el dinero. Efectivamente, el nodo central de la red emite una moneda, llamada crédito, a una cantidad fijadeLight (1972Light ( , 1988Light ( , 2000), EdnaBonacich (1988 Bonacich ( , 2000 ) y AlejandroPortes (1985Portes ( ,1986Portes ( ,1987Portes ( ,1994grandes empresas, de ahí la expresión " minorías intermediarias " , las cuales pueden emplear también a trabajadores de otras etnias. Se trata, de nuevo, de un mercado secundario de trabajo, pero segmentado étnicamente. ... Invitación a la Antropolog'ia Económica. Book Full-text available Jan 2007 José Luis Molina Hugo Valenzuela-Garcia ... trueque en la actualidad, es el dinero. Efectivamente, el nodo central de la red emite una moneda, llamada crédito, a una cantidad fijadeLight (1972Light ( , 1988Light ( , 2000), EdnaBonacich (1988 Bonacich ( , 2000 ) y AlejandroPortes (1985Portes ( ,1986Portes ( ,1987Portes ( ,1994grandes empresas, de ahí la expresión " minorías intermediarias " , las cuales pueden emplear también a trabajadores de otras etnias. Se trata, de nuevo, de un mercado secundario de trabajo, pero segmentado étnicamente. ... Economic Anthropology Chapter Full-text available Jan 2016 Hugo Valenzuela-Garcia José Luis Molina ... Enfin, la migration se distingue par une surreprésentation dans les ateliers de confection, qui sont l'une des trois modalités de la production de vêtements, avec la fabrication en usine et à domicile. L'ensemble de ce dispositif productif fonctionne en complémentarité, grâce aux mécanismes complexes de la sous-traitance (Bonacich et Appelbaum, 2000 ; Green, 2003 ;Leite, 2004 ;Silva, 2008). Les années 1980-1990 ont vu l'activité en usine reculer, au bénéfice de la production en atelier (Pochmann, 2004) et à domicile (Buechler, 2014). ... Production et usage des ressources urbaines en migration : les ateliers de confection à São Paulo Article Full-text available Sylvain Souchaud This article aims to analyze the production and access to urban resources in a context of migration. Urban resources are treated as devices and practices that are constitutive of the urban environment. To characterize them, we target groups of South American migrants, workers and small employers inserted into the sweatshop economy. We will see how, in a metropolitan context where international migrants face difficulties entering the labor market and access to housing, workshops are organized by re-using urban fabric elements, namely certain frame configurations, and produce their own supply of housing to labor, to guarantee its cheapness and productivity. Therefore, workshops system is a user as much as a producer of specific urban resources. ... 58 A small segment of these outmigrants are currently pursuing higher education degrees in La Universidad Autónoma de Chapingo in Mexico City (7 men and 1 woman) and COBACH in Comitán, Chiapas (2 women). 59 For a review of the rampant inequalities underpinning sweatshop labor in Los Angeles, please read, Bonacich and Appelbaum 2000. expressed how "piece-work is a Korean invention". ... Migration Politics and Human Rights Article Full-text available Apr 2007 Óscar Gil-García Forced migration of Guatemalans and their participation in the labor markets of Mexico and the US has led to their categorization as economic migrants. This identification loses sight of the contextual experience of forced migration for more than economic reasons. My research methods apply a cultural analysis that blends feminist ethnography with photography. By distributing single-use color cameras, participants’ have been able to use a visual technological tool in the field and record aspects of their lives of greatest concern. My use of a feminist ethnographic approach aims to challenge the dominant representation of migrants, based on a heteropatriarchical gendered script, which defines women as domestics and nurturer’s of children, while men are viewed as mobile wage earners. This gendered construction of women-as- domestic has become a dominant photographic method in recording forced migrants by International Humanitarian Organizations such as the U.N. High Commissioner for Refugees that reinforce their visual cultural representation as technologically backward that has direct material consequences in the global feminization of the labor force. The photographs in my study, and participants’ interpretations of them, enhance globalization/gender theories by capturing the cultural practices and technological forms that enable or prevent women or men from participating in particular forms of production and exchange. ... Western contexts have highlighted the role that immigrant labour played in maintaining the supply of low cost labour (Phizacklea 1990; Bonacich and Appelbaum 2000) , the case of Vietnamese clothing workers migrating to Malaysia takes shape via the same kind of state-organised systems of temporary, short-term contact labour migration noted in Elias's chapter on domestic worker migration (this volume). The globalisation of the household is a transformation that potentially offsets the difficulties of forming households with inadequate financial means and/or home based care. ... Vietnamese Migrant Clothing Workers in Malaysia: global Production, Transnational Labour Migration and Social Reproduction Chapter Full-text available Jan 2013 Vicki Crinis The household in Southeast Asia is a site that provides the labour necessary for global production. And yet, under conditions of global restructuring many households have become worse off because of the way in which new employment opportunities for women have frequently been concentrated in labour-intensive, low-paid, feminized sectors of the economy. For transnational labour migrants, especially women from poor households, globalization has been experienced in terms of reconfigured household survival strategies. Globalization does not appear to offer much in the way of ‘empowerment’. This chapter discusses unskilled Vietnamese migrant workers in the Malaysian clothing industry. The chapter points to how social relations of reproduction are being transformed through capital accumulation, neoliberal trade policies and transnational labour migration. ... For example, Apple recently came into the limelight for various 'unfair labour' practices at its Chinese factories, including the use of under-age labour, improper disposal of hazardous waste, and excessive overtime being forced on its workers (Nuttall 2012). While such conditions are often depicted by many industrialists and journalists as aberrations from the norms and standards of international labour law, anthropologists have long shown how these are far from abnormal and, indeed, often fundamental to the workings of Western capitalism (Nash 1979; Taussig 1980; Ong 1987; Bonacich and Appelbaum 2000; Ingraham 1999). These works provide a useful starting point for refl ecting on the wider question of what perspectives from the periphery have to offer the discipline. ... Up Close and Personal Intro & Conclusion Data Full-text available Apr 2016 Cris Shore Susanna Trnka ... For example, Apple recently came into the limelight for various 'unfair labour' practices at its Chinese factories, including the use of under-age labour, improper disposal of hazardous waste, and excessive overtime being forced on its workers (Nuttall 2012). While such conditions are often depicted by many industrialists and journalists as aberrations from the norms and standards of international labour law, anthropologists have long shown how these are far from abnormal and, indeed, often fundamental to the workings of Western capitalism (Nash 1979; Taussig 1980; Ong 1987; Bonacich and Appelbaum 2000; Ingraham 1999). These works provide a useful starting point for refl ecting on the wider question of what perspectives from the periphery have to offer the discipline. ... Preface: Anthropologists up close and personal Article Full-text available Jan 2013 Cris Shore Susanna Trnka Between Exploitation and Repression: The Immigration Industrial Complex and Militarized Migration Management Chapter Jan 2022 David B. Feldman This chapter locates the emergence of an immigration industrial complex profiting from the detention and deportation of precarious migrants within the development and historical trajectory of distinct migration regimes in the United States. On the one hand, the IIC emerged as an integral component of a migration regime centered around the illegalization and criminalization of low-wage migrant and immigrant workers during the era of neoliberal capitalist globalization. On the other hand, the IIC undermines this very regulatory regime by systematically removing such workers from the labor market. I consider the possibility that this contradiction may be partially resolved through transition to a nascent regime of militarized migration management in which the state plays a more active role in the formal regulation of precarious migrants by promoting guestworker programs and various legalization schemes for certain undocumented immigrants, while denying entry to surplus populations with highly securitized borders.KeywordsImmigration industrial complexMilitarized migration managementMigration regimeAccumulation by repressionMigrant labor Global socioeconomic networks of the apparel industry: the case of the Los Angeles Fashion District Article Full-text available Jun 2022 GeoJournal Su-Jeong Kim This study aims to identify the Fashion District as the point where the expansion of the global network meets a local context and to investigate the influence of the apparel industry’s global network on the spatial change of the Los Angeles Fashion District. Los Angeles apparel industry is expanding and restructuring its production network and global value chain through the alliance of various interest groups in the neoliberal system. The garment industry in the United States and Los Angeles has been heavily influenced by international institutions such as the multi-fiber arrangement. As the global production geography is reorganized and expanded due to changes in international systems and labor costs, the pan-pacific global value chain of China, Southeast Asia, and Central and South America, centering on the Los Angeles Fashion District is formed and functioned. In the business relationship between the firms in the production process, not only the cost but also the ethnicity of the subjects in charge of each production process tended to play an important role. Especially in the case of the Los Angeles apparel industry, the connectivity between Koreans located at each stage of production is emphasized. Korean manufacturers prefer Asian subcontractors rather than geographically far closer Latin American countries because of the cost of labor and the cost of establishing factories and the policies of the national and city governments, as well as socio-cultural characteristics such as co-ethnic relations. The Subversive Stitch Revisited Chapter Jul 2020 Lisa Vinebaum This chapter proposes a substantial shift away from understandings of sewing as a gendered, individual, domestic activity, theorized most notably by Rozsika Parker in her 1984 book, The Subversive Stitch: Embroidery and the Making of the Feminine. While acknowledging Parker's important contributions to the study of sewing by women in Europe and Britain, it reflects critically on her lack of attention to class, race, and geography. The chapter proposes new and more inclusive understandings of the subversive stitch, considered through projects by artists Aram Han Sifuentes and Carole Frances Lung. Han Sifuentes uses sewing to call attention to discrimination against immigrants, and to help alleviate the isolation many experience. She sees the US Citizenship Test Sampler as a project that builds community among increasingly vulnerable populations of noncitizens in the US. Today's subversive stitches are just as likely to be sewn in public by groups of people sewing together. Predatory Purchasing Practices in Global Supply Chains and the Employment Relations Squeeze in the Indian Garment Export Sector Article Aug 2019 Mark S. Anner Worker rights abuses in garment global supply chains have long been acknowledged in the literature (Barrientos et al., 2003; Bonacich and Appelbaum, 2000). What has been less explored is the recent growth in certain decent work deficits and its causes. This article links this growth to predatory purchasing practices tied to growing power asymmetries. As illustrated through the case study of the garment export sector in India, these predatory practices have resulted in downward pressure not only on wages, but also an increase in non‐standard work and greater verbal and physical abuse tied to increased productivity demands. Addressing these practices requires transforming purchasing practices through pricing mechanisms that cover the cost of decent work, appropriate state regulation, and worker involvement. Migration and Refugees Chapter Aug 2017 Cecilia Menjívar With increasing trends of globalization, the gendered nature of migration has been accentuated. Current world systemic political and economic dislocations generate large population movements, most of these punctuated by gender differences. Women in different areas of the world, mainly in Asia, the Caribbean, and Latin America, migrate internally to seek jobs in the ever‐increasing export‐processing zones in their own countries. Sassen‐Koob (1984) observes that these women, through their work in export processing zones, become familiar with the different lifestyles and consumer practices of the countries where investment capital originates. Thus, when they are laid off from the factories (where the turnover rate is very high), they do not go back to the rural areas from where they originated (where their social and economic reinsertion will be difficult), but instead become part of an increasing pool of women who migrate internationally in search of jobs (as domestics or caring for the increasingly ageing population of richer countries, etc.) in the wealthier countries that house the firms that invest in the export‐processing zones. Men also participate in large migratory movements, but given the jobs that are available in today's world economy, it is women's migration that has increased both internally and internationally. Thus, men migrate under organized labor recruitment programs, such as those that exist between the Philippines and Gulf countries or between Mexico and the United States. But relatively more women migrate as workers under these recruitment programs, or as family members, while relatively fewer move on their own or independently from these institutionalized practices. The matrix of exploitation and temporary employment: Earnings inequality among Inland Southern California's blue‐collar warehouse workers Article Nov 2018 Juliann Emmons Allison Joel S. Herrera Jason Struna Ellen Reese The “just in time” neoliberal global consumer economy has given rise to logistics or “goods movement” hubs, reshaping regional labor markets near port cities, and increasing the demand for temporary blue‐collar workers. This article provides a case study of earnings inequality among nonsupervisory warehouse workers in Inland Southern California, most of whom are Latina/o. Combining insights from labor market segmentation and intersectional feminist theories, we argue that employer discrimination against temps has become a significant axis of workplace inequality that interacts with other social inequalities to segment workers into lower and higher paying positions within a complex “matrix of exploitation.” Our statistical analysis uses data from an original survey of workers as well as the American Community Survey to analyze wages, income, and occupational segregation among Inland Southern California's blue‐collar warehouse workforce. We find that, controlling for other factors, temporary employment status negatively affects hourly wages and annual earnings. Consistent with the expectations of intersectional feminist theory, the hourly wages of immigrant Latinas, and the annual incomes of immigrant Latinas who are naturalized citizens, were significantly lower than those of all other warehouse workers, controlling for the effects of other factors. Immigrant women were also disproportionately employed as temps and in the lowest paid occupation. Liabilities and Benefits Associated with the Involvement of Undocumented Immigrants in Informal Entrepreneurship in the US: Future Research Paradigms for Creating Innovative Business Activity Chapter Jan 2019 Steven J Gold The many undocumented immigrants present in the US, together with the growing popularity of entrepreneurship as a job-creation strategy, have enhanced both the number and acceptance of self-employed undocumented immigrants in US society. This article draws upon multiple sources of data including literature review, official statistics, journalism and the author’s own ethnographic research to assess the impact of undocumented immigrants’ growing involvement in informal entrepreneurship. It considers the extent to which self-employment can provide income for undocumented immigrants generally and further seeks to determine rates of entrepreneurship in relation to migrants’ demographic characteristics, socioeconomic status, gender, nationality, location, and realm of economic specialization. Finally, the paper reviews the social, political and economic impact of undocumented immigrants’ entrepreneurship on the larger society. Local Sweatshops in the Global Economy: Accumulation Dynamics and the Manufacturing of a Reserve Army Chapter Full-text available Apr 2018 Jerónimo Montero Bressán Sweatshops lie at the core of the fashion industry worldwide. While awareness about large sweatshops in export-processing zones is broad, little is known about small local sweatshops in large cities both in core and peripheral economies, employing migrant labour and supplying cheap garment to fast-fashion branded retailers. Having been left at the margins of this industry during Fordism, these ‘local sweatshops’ are back since the late 1970s. In these, working conditions range from precarious employment to forced labour. This chapter asks what does the return of local sweatshops mean for debates on unfree labour and capitalist accumulation. Through an analysis of the changes in the production and commercialisation of fashion clothing since the late 1970s, I show that the flourishing of forced labour during recent decades along the success of well-known brands and retailers, suggests that, far from being a pre-capitalist reminiscence, forced labour is not only compatible with capitalist accumulation, but it can also be critical for its survival. Following from this, responses from unions and community organisations are analysed, based on the case of anti-sweatshop activism in Buenos Aires. I conclude by showing that when the agenda against forced labour is taken by NGOs rather than by labour activists, class perspectives are largely absent and improvements are poor. 2. Voluntary Versus Binding Forms of Regulation in Global Production Networks: Exploring the “Paradoxes of Partnership” in the European Anti-Sweatshop Movement: Health and Safety of the World's Garment Workers Chapter Full-text available Jan 2017 Florence Palpacuer Beyond disciplinary boundaries: Leveraging complementary perspectives on global labour Article Full-text available Jul 2017 Tobias Schulze-Cleven Gary Herrigel Nelson Lichtenstein Gay Seidman This symposium-style article brings together scholars from history, sociology and political science to explore how different disciplinary traditions can contribute to a productive dialogue on workers’ collective action and labour power around the world. Grounded in reflections on recent research in three disciplinary communities, the article encourages scholars to tap into findings from other academic traditions to refine the focus and the contextualization of their own analyses. This strategy of moving beyond disciplinary boundaries, the article argues, promises to expand inherited styles of inquiry by encouraging analyses with a wider selection of cases, a more conscious temporal anchoring and broadened geographic reach. The evolution of scholarship along these lines would honour each discipline’s particular conceptual commitments and simultaneously seek to enlist them more broadly for a deeper understanding of labour’s contemporary reorientation. View Monitoring Workers’ Rights: The Limits of Voluntary Social Compliance Initiatives in Labor Repressive Regimes Article May 2017 Mark S. Anner Since the early 1990s, a range of corporate monitoring and multi-stakeholder initiatives have sought to address the violation of workers’ rights by monitoring suppliers in global supply chain and attempting to remediate violations. However, their effectiveness in the area of freedom of association rights has been limited, particularly in labor-repressive regimes. This is because these initiatives have pursued inadequate strategies and lack either traditional forms of state power, notably the ability to sanction violators, or the leverage provided by activist campaigns. Recent social compliance program approaches, such as forming of worker-management committees, are largely of limited effectiveness in regimes in which workers face employer or state controlled unions. And most production in light industry supply chains such as apparel takes place in labor repressive regimes. Has the Suburbanization of Ethnic Economies Created New Opportunities for Income Attainment?*: Suburbanization of Ethnic Economies Article Feb 2017 SOC SCI QUART Mahesh Somashekhar Objective International migration to the U.S. suburbs has upended many theories of urban inequality and immigrant incorporation, including ethnic economy theory. This article is the most comprehensive study conducted to date on the reasons behind ethnic economy suburbanization and its effect on earnings. Methods The article uses regression techniques to analyze Census microdata from 1990 to 2010. A series of analyses that aggregate and disaggregate trends across nine ethnic groups identify the extent and influence of ethnic economies in suburban areas. Results Ethnic economy suburbanization is strongly associated with ethnic residential suburbanization, and earnings are no different in the suburban and urban portions of the ethnic economy. Conclusion Although existing research highlights the uniqueness of suburban ethnic economies, suburban ethnic economies are delivering outcomes similar to those found in urban areas. This supports the body of literature arguing that differences between immigrant incorporation patterns in cities and suburbs are diminishing. Apple, Foxconn, and China’s New Working Class Chapter Full-text available Jan 2016 Jenny Chan Ngai Pun Mark Selden Jenny Chan, Ngai Pun and Mark Selden. 2016. “Apple, Foxconn, and China’s New Working Class.” Pp. 173-89 in Achieving Workers’ Rights in the Global Economy, edited by Richard P. Appelbaum and Nelson Lichtenstein. Ithaca, NY: Cornell University Press. Organizing Temporary, Subcontracted, and Immigrant Workers: Lessons from Change to Wins Warehouse Workers United Campaign Article Aug 2016 Juan De Lara Ellen Reese Jason Struna Since 2008, Warehouse Workers United (WWU) has organized thousands of low-wage warehouse workers in Southern California’s Riverside and San Bernardino Counties, many of whom are temporary, subcontracted, and immigrant workers. Warehouse Workers Resource Center (WWRC), formed in 2011, has provided additional legal services and other resources to warehouse workers. Combining protest tactics, a legal and media strategy, and a commodity chain organizing strategy, WWU and WWRC helped warehouse workers to win back millions of dollars of stolen wages and to pass new regulatory legislation for employers of warehouse workers. In coalition with other labor organizations, they also obtained an agreement by Walmart to improve its workplace safety standards. This case study, based on field research and interviews with key informants, provides important lessons for those seeking to organize marginalized workers in other industries and regions. Sweatshop Workers in Buenos Aires: The Political Economy of Human Trafficking in a Peripheral Country Chapter Full-text available Jan 2015 Jerónimo Montero Bressán Eliana Ferradás Abalo In March 2006, a fire broke out in a medium-sized garment workshop in the working-class Caballito neighbourhood of Buenos Aires, Argentina, leading to the death of two Bolivian workers and four children. All of them were living in the place with another 60 people. They could not escape from the sweatshop because the doors were locked. The tragedy triggered the disclosure of thousands of ‘local sweatshops’ (Montero y Arcos, unpublished) that supply small, medium and large local and international brands. In 2010, the Under-Secretariat of Labour calculated that in Buenos Aires city alone there were about 5,000 sweatshops (Lieutier, 2010), and at least double that number in Greater Buenos Aires.1 Factory Women under Globalization: Incorporating Japanese Women into the Global Factory Debate Chapter Jan 2005 Mayumi Murayama Women working in factories have been a topic of heated debate since the time of the first Industrial Revolution in Britain (Engels, 1845). In the countries that followed Britain, such as France and the USA, women again played a crucial role as industrial labourers, contributing to the successful transformation of the national economy as well as to their households’ livelihoods (Tilly and Scott, 1978; Dublin, 1979; Moran, 2002). Japan was no exception. Since the establishment of the first modern silk reeling mill in 1872 and during the subsequent era of the industrial revolution, female workers constituted the majority of the industrial workforce (Hosoi, 1954; Yamamoto, 1977; Nakamura, 1985; Tsurumi, 1990; Miyake, 1991; Molony, 1991). See Murayama (2003) for a detailed bibliography. “Most Overrated Western Virtue”: The Politics of Knowledge in Zadie Smith’s White Teeth Chapter Jan 2007 Grace Kyungwon Hong A rchie Jones, one of the many hapless “heroes” of Zadie Smiths novel White Teeth (2000)1, faces a decision of epic proportions. As a seventeen-year-old English soldier during World War II, he finds himself alone in the Bulgarian woods with perhaps the most iconic representation of twentieth-century evil: a Nazi collaborationist doctor whose expertise in genetics and whose participation in the “internal German matters” regarding “the sterilization program” and the “euthanasia policy” (WT , 90), hints at the darkest and most horrifying kinds of human experimentation. Should Archie kill this man, this representative of the amoral nadir of human existence, a man who routinely and casually decided who should live and who should die? Yet in so doing, Archie would become the very thing he is destroying. Should he let him go, and allow this man to further perpetuate his kind of eugenicist science, in fact, allow him to turn a strain of eugenics into the defining science of the twentieth century? One might think that this decision upon which, one could argue, the fate of the world hangs, would be difficult, if not nearly impossible to make. But it is not so very hard for Archie, or at least, it is not so hard for long. Because unlike you or me, Archie does not worry himself with any of these grand ideas. Instead, to make this decision on which, one could argue, the entire fate of the twentieth century hinges, Archie flips a coin. And he flips it badly. Challenges to Authority in Global Politics Chapter Jan 2008 Janie Leatherman The al-Qaeda attack on the United States on September 11, 2001, was a crime against humanity and a challenge to U.S. hegemonic leadership and the entire post—World War II political order. Although the “war on terror” has replaced the once-familiar Cold War confrontation between communism and capitalism, the discourse and the power behind the war on terror mask a larger political process underfoot in global politics. Thus, this volume seeks to unsettle the predominance of the war on terror lens through which much of the post—Cold War international relations have been cast since September 11. There are many actors on the global stage competing for power and authority—some of them using punitive means, others consensual. Global capitalism, global civil society, global governance institutions and organizations, and a wide range of “illicit” networks—ranging from al-Qaeda to drug smugglers and human traffickers—present a bewildering array of stakeholders that variously confront and collaborate with one another using different strategies of power. Among the questions that must be asked are, on this crowded global stage, who is in charge of what, how do they stay in charge, and what are the effects of their strategic maneuvers? How do Migrant Workers Respond to Labour Abuses in “Local Sweatshops”? Article Jun 2016 Jerónimo Montero Bressán Ayelén Arcos This article aims to provide empirical evidence on understanding how migrant workers’ responses to labour exploitation in low-wage economies are articulated. Inspired by the low levels of conflict among workers in small urban sweatshops in Italy and Argentina, we ask ourselves what contextual and subjective factors prevent workers from organising collectively. Here we argue that in order to understand the nature of their responses, it is necessary to consider not only the organisation of the labour process, but also the class divisions within migrant communities. We also bring in briefly the role of the state in (mis)regulating migrant labour exploitation. We conclude by showing that workers’ responses are highly individualised and that community leaders with economic interests in sweatshop economies may play a role in securing their continuation by channelling the workers’ responses towards the defence of the “ethnic economy”. Why Place Matters: An Introduction Chapter Jul 2016 Dana Collins This introductory chapter begins with a conceptualization of the power of urban place, as the author conducted a thirteen-year urban ethnography in a former sex and current tourism district, Malate, in the City of Manila, the Philippines. The author explores the place in the academic literature and offers a methodological overview of the power and politics of studying a sexual community in the global South. By examining the global urbanization, place, and gay urban communities scholarship, this chapter explains how place is a factor in neighborhood change. The reader is introduced to a core theme in the book—how powerful and marginal urban actors, in the face global homogenization, draw from place for urban renewal, and how sexuality became central to urban change in Malate. Global Production in a Romanian Village: Middle-Income Economy, Industrial Dislocation and the Reserve Army of Labor Feb 2016 Milosz Miszczynski This article contributes to the discussion about the process of relocation of industrial production. The study focuses on a community in Eastern Europe which witnessed a cycle of events: from localization of a high-scale global factory in 2007 to its unexpected displacement in 2012. In this study is analyzed the process of the community’s inclusion in the production system, and it is argued that the challenge of production mobility is distinctively different in middle-income economies. Employing a cultural approach to the study of global production, the process of social reconstruction caused by the investment is chronologically outlined and its outcomes for the local labor market and host community are described. The State, Unauthorized Mexican Migration, and Vulnerability in the Workplace Feb 2016 Jacqueline Olvera Die strukturelle Krise der flexibilisierten Arbeit: Strategien und Aussichten für die transnationale gewerkschaftliche Organisierung in der Bekleidungs- und Sportschuhbranche Jan 2011 Jeroen Merk Verhaltenskodizes und Sozialaudits – was haben sie den Beschäftigten der Bekleidungsindustrie in den Produktionsländern gebracht? Book Nov 2011 Jeroen Merk Unions and the Unionization of Latinas/os in the United States Chapter Jan 2008 Héctor L. Delgado This chapter will focus on the participation of Latina/o workers in past and contemporary labor struggles and the role that Latina/o workers can, should, and are likely to play in the labor movement in the 21st century. The emphasis, because it is the emphasis in the literature, will be on Mexican workers in the West and Southwest and, to a lesser degree, Puerto Rican workers on the East Coast. However, one of the aims of this chapter is to call attention to other Latina/o groups in the labor movement and to underscore the need for future research on these groups and other regions of the country, including the South, long a part of the country with very low levels of unionization. This chapter will provide a review of important labor campaigns in which Latina/o workers, often immigrant workers, played a critical role. Latina/o workers’ militancy revealed in this review belies stereotypes of the passive Latina/o and calls into question the claim by some in the labor movement that these workers were (and are) “unorganizable.” The chapter also offers contemporary evidence of Latina/o workers’ receptivity to unionization and the benefits of unionization for workers, including Latina/o workers, the group likely to benefit the most from a revitalized labor movement. Organized labor’s woes are discussed, as is its future and the future of Latina/o workers. Finally, this chapter prescribes what organized labor needs to do to organize Latina/o workers, including recognizing the heterogeneity of this population and the implications of this heterogeneity for organization, and what scholars can do to enhance our understanding of the evolving relationship between organized labor and Latinas/os. Article Edna Bonacich and Richard P. Appelbaum, Behind the Label: Inequality in the Los Angeles Apparel Indu... January 2002 · International Labor and Working-Class History Jefferson Cowie	https://www.researchgate.net/publication/248862559_Behind_the_Label_Inequality_in_the_Los_Angeles_Apparel_Industry
OBM Genetics \| Newborn Screening for Genetic Diseases: An Overview of Current and Future Applications Newborn screening (NBS) for inborn errors of metabolism (IEM) was introduced more than 50 years ago with the testing of phenylketonuria (PKU) using blood spots deposited on a filter paper after heel prick. NBS aims to identify early after birth inherited disorders for which clinical management and pre-symptomatic treatment will significantly decrease morbidity and mortality. While NBS for a few other disorders was implemented in some specific jurisdictions over the following decades, it is with the introduction of tandem mass spectrometry (MSMS) by the end of the 1990’s that NBS expansion really took place. MSMS has had a tremendous impact on NBS, but led to heterogeneity in NBS disorders panel between jurisdictions that have introduced new conditions at various rates using different decision-making processes. In addition to disorders tested by MSMS, many programs have included other inherited disorders using various testing methodologies, including cystic fibrosis (CF), sickle cell diseases (SCD), while some others have been recently introduced in some jurisdictions or are under considerations, such as severe combined immunodeficiency (SCID), lysosomal storage disorders and X-linked adrenoleukodystrophy (X-ALD). As the cost of DNA testing decreases, the future of NBS will inevitably include genome sequencing, which will represent an opportunity to detect, treat and prevent more serious early-onset health conditions in the best interest of newborns, provided that public health authorities rule on ethical and policy issues, such as the identification of variant of unknown significance and the disclosure of incidental findings. Finally, despite the current exciting technological advances in the field of NBS in the Northern hemisphere, we should not be blinded that despite various initiatives, about two-thirds of neonates born in low-resource areas are still waiting to have access to NBS. OBM Genetics (ISSN 2577-5790) Journal Flyer OBM Geneticsis an internationalOpen Accessjournal published quarterly online by LIDSEN Publishing Inc. It accepts papers addressing basic and medical aspects of genetics and epigenetics and also ethical, legal and social issues. Coverage includes clinical, developmental, diagnostic, evolutionary, genomic, mitochondrial, molecular, oncological, population and reproductive aspects. It publishes research articles, reviews, communications and technical notes, etc. There is no restriction on the length of the papers and we encourage scientists to publish their results in as much detail as possible. Indexing: COPE DOAJ-Directory of Open Access Journals Scopus Google Scholar Archiving:full-text archived in CLOCKSS. Rapid publication:manuscripts are undertaken in 15.0 daysfrom acceptance to publication (median values for papers published in this journal in the second half of 2021, 1-2 days of FREE language polishingtime is also included in this period). Open Access Review Newborn Screening for Genetic Diseases: An Overview of Current and Future Applications Damien Bouvier 1, Yves Giguère 2, 3, * 1. Biochemistry and Molecular Genetic Department, CHU Clermont-Ferrand, Clermont-Ferrand, France, and Université Clermont Auvergne, Faculty of medicine, CNRS 6293, INSERM 1103, GReD, Clermont-Ferrand, France 2. Centre de recherche du CHU de Québec-Université Laval, Québec City, Canada 3. Department of Molecular Biology, Medical biochemistry and Pathology, Faculty of Medicine, Université Laval, Québec City, Canada *Correspondence:Yves Giguère Special Issue:Genetic Screening Received:July 01, 2019 \|Accepted:September 04, 2019 \|Published:September 06, 2019 OBM Genetics2019, Volume 3, Issue 3, doi:10.21926/obm.genet.1903093 Recommended citation:Bouvier D, Giguère Y. Newborn Screening for Genetic Diseases: An Overview of Current and Future Applications. OBM Genetics2019; 3(3): 093; doi:10.21926/obm.genet.1903093. © 2019 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited. Abstract Newborn screening (NBS) for inborn errors of metabolism (IEM) was introduced more than 50 years ago with the testing of phenylketonuria (PKU) using blood spots deposited on a filter paper after heel prick. NBS aims to identify early after birth inherited disorders for which clinical management and pre-symptomatic treatment will significantly decrease morbidity and mortality. While NBS for a few other disorders was implemented in some specific jurisdictions over the following decades, it is with the introduction of tandem mass spectrometry (MSMS) by the end of the 1990’s that NBS expansion really took place. MSMS has had a tremendous impact on NBS, but led to heterogeneity in NBS disorders panel between jurisdictions that have introduced new conditions at various rates using different decision-making processes. In addition to disorders tested by MSMS, many programs have included other inherited disorders using various testing methodologies, including cystic fibrosis (CF), sickle cell diseases (SCD), while some others have been recently introduced in some jurisdictions or are under considerations, such as severe combined immunodeficiency (SCID), lysosomal storage disorders and X-linked adrenoleukodystrophy (X-ALD). As the cost of DNA testing decreases, the future of NBS will inevitably include genome sequencing, which will represent an opportunity to detect, treat and prevent more serious early-onset health conditions in the best interest of newborns, provided that public health authorities rule on ethical and policy issues, such as the identification of variant of unknown significance and the disclosure of incidental findings. Finally, despite the current exciting technological advances in the field of NBS in the Northern hemisphere, we should not be blinded that despite various initiatives, about two-thirds of neonates born in low-resource areas are still waiting to have access to NBS. Keywords Newborn screening; genetic diseases; next-generation sequencing; low-resource areas 1. Newborn Screening Then and Now: An Overview In 1961, Dr Robert Guthrie described a method of testing newborns for Phenylketonuria (PKU). The first disorder screened for, PKU, is a rare, recessively inherited enzyme deficiency of the phenylalanine hydroxylase that generally results in severe mental retardation unless treated from early infancy [ 1]. Dr Guthrie proposed a simple blood sampling method that involved phenylalanine measurement from heelstick blood samples dried on a filter paper. The advantages of dried blood spots on filter paper are that sample collection is easier and that logistic aspects related to sample storage and shipment through postal services to a central laboratory for testing can be relatively limited, without the need of a refrigerator or dry ice [ 2]. Rapidly, US states collaborated to the implementation of universal screening, and already in 1963 over 400 000 newborns from 29 states were screened, which revealed 37 cases of PKU. All affected children were on low phenylalanine dietary treatment by the age of 1 month, and the feasibility of mass screening was clearly shown [ 3]. By 1965, 32 states performed newborn screening (NBS) in the USA, including 27 states enforcing mandatory screening [ 4]. In the following years, many other countries from the western world implemented newborn screening for PKU. The next breakthrough in newborn screening came in 1975 when Jean Dussault published a method of newborn screening for congenital hypothyroidism (CH) using the same filter paper in use for PKU [ 5]. Most developed countries had adopted screening for PKU and CH in the 1970’s. In the next two decades, there were a very few initiatives to add other disorders to NBS programs, such as congenital adrenal hyperplasia (CAH), haemoglobinopathies, biotinidase deficiency and cystic fibrosis (CF) [ 6]. Some of the additional disorders added to the limited NBS panel included those for which there was a high prevalence in the related jurisdiction. As an example, NBS for hereditary tyrosinemia type 1 (HT1) due to fumarylacetoacetate hydroxylase deficiency, was added in 1970 in Québec where a genetic founder effect in the French-Canadian population led to an increased prevalence of the disorder [ 7]. HT1 was first screened by tyrosine measurement, but eventually succinylacetone, a byproduct, which showed to be more sensitive and specific and became the 1 sttier screening test for HT1. In the 1990s the development of electro-spray ionisation tandem mass spectrometry (MSMS), a multiplex platform technology allowing multianalyte screening from one blood spot, and thus the simultaneous detection of many rare inherited disorder after only one single injection into the instrument, led to the massive expansion of NBS [ 8]. Since then, many jurisdictions have progressively performed their own decision-making process for the expansion of NBS, which led to heterogeneous NBS disorder panels. Some countries adopted a very conservative approach including only a few disorders, while others have increased significantly the number of conditions screened to more than 30 in their NBS panel. To add to the complexity, some countries such as the United States and Canada do not have nationally mandated NBS programs, as they fall under territorial (states, provinces,…) jurisdictions, each of which may follow their own processes of NBS expansion. In the United States, however, to decrease state-to-state heterogeneity between NBS disorders panels, which raised equity concerns of advocacy groups [ 9], the Secretary of Health and Human Services’ Advisory Committee on Heritable Disorders in Newborns and Children (SACHDNC) mandated the American College of Medical Genetics to provide the Secretary with national NBS recommendations for a Recommended Uniform Screening Panel (RUSP): out of 88 conditions assessed, this led to the adoption of a core of 29 conditions to be screened and 25 secondary target conditions [ 10]. The adoption of the RUSP in the United States led to significant harmonization, although the methodology used was debated [ 11, 12]. Since the initial version, other conditions, such as severe combined immunodeficiency disease (SCID) [ 13] [ 14], X-linked adrenoleukodystrophy, critical congenital heart disease (CCHD)[ 15](performed by pulse oximetry), Pompe disease, and Mucopolysaccharidosis type I (MPS I), as well as spinal muscular atrophy due to homozygous deletion of exon 7 in SMN1 and newborn hearing screening [ 16] have been included in the core conditions of the RUSP, which now represents 35 conditions [ 17]. Of note, screening techniques for lysosomal diseases are also rapidly developing. A tandem mass spectrometry assay in which the enzymatic activities of 9 lysosomal enzymes (α-glucosidase, α-galactosidase A, α-I-iduronidase, acid sphingomyelinase, acid β-glucocerebrosidase, galactocerebrosidase, iduronate-2-sulfatase, N-acetyl-galactosamine-6-sulfatase, N-acetyl-galactosamine-4-sulfatase) was developed to screen Pompe disease, Fabry disease, Hurler disease, Niemann-Pick-A/B disease, Gaucher disease, Krabbe disease, mucopolysaccharidosis II, IVA and VI, respectively [ 18]. Peroxisomal diseases as X-linked adrenoleukodystrophy are also studied using LC-MSMS [ 19]. Some conditions, such as Duchenne muscular dystrophy are not included in the core conditions of the RUSP but are screened in newborns in some jurisdictions [ 20]. Approved treatments for Duchenne muscular dystrophy may change the natural history of this disease [ 21]. To avoid diagnostic delay and provide optimal treatments, early identification of patients through NBS will be necessary. If the ACMG recommendations had some influence in the decision-making process outside the United States, they did not receive worldwide support, most probably because different jurisdictions evaluate relevance of conditions to be introduced in their NBS programs using different angles, which leads to various interpretations of similar criteria. In their 1968 World Health Organization report, Wilson and Jungner identified 10 principles and practice of population screening to guide the selection of conditions that would be suitable for screening (Table 1) [ 22]. Population screening aims to identify disorders that fulfill those criteria that are mainly based on the severity and natural history of the disorder, the capacity to detect the condition at an early pre-symptomatic stage using an acceptable test, and the availability of an acceptable treatment. Also, they considered the impact of screening on the cost of case-finding, which should be economically balanced in relation to medical costs. Considering the many technological advances that were introduced since, which made screening a topic of growing importance and controversy, an updated WHO report was published forty years after the classic Wilson and Jungner criteria were outlined [ 23]. The WHO criteria were not specifically directed for genetic application and newborn screening, but they have stood the test of time. While there is a broad consensus on these population-based screening criteria, there is variability in how they are interpreted. As mentioned by Wilcken and Wiley regarding NBS, there is in fact little hard evidence about the benefits of screening [ 6], which probably explains, as least for a significant part, the differences in the evaluation of the relevance of conditions to be screened between the different territories [ 24]. One must not only consider the benefits, but also the potential harms for each condition added to a NBS panel, namely the increase of false positive results leading to unneeded interventions, as well as the potential parental psychological distress and potential distortion of parental perception about the child, while expanded screening may give rise to so called “patient in waiting”, i.e. individuals with a genetic diagnosis who remain asymptomatic for years or decades [ 25]. After performing a systematic review of the literature on NBS criteria worldwide, Jansen et al.concluded that despite commonalities, NBS programs are using different approaches to explore the same policy issues, including: the beneficiary of NBS, definition of criteria, the way conditions are assessed, level of evidence required, and recommendations after assessment, which potentially results in increased disparity across NBS programs internationally [ 26]. Table 1Summary of the 1968 Wilson and Jungner criteria for screening, [ 22] (updated by Andermann et al.in 2008 [ 23]). Table 1Summary of the 1968 Wilson and Jungner criteria for screening, [ 22] (updated by Andermann et al.in 2008 [ 23]). Principal Wilson and Jungner Criteria for Screening• Condition is an important health problem• Acceptable treatment available• Facilities for diagnosis and treatment available• Knowledge of the natural history of the disorder • Presymptomatic intervention improves outcomes • Simple and low cost test with excellent performance • Resources available for follow-up of positive individuals • Program acceptable to the population • Development of a program policy • Cost of case-finding is acceptable 2. Next Generation Sequencing in the Field of NBS: Potential and Concerns With the recent sequencing of the entire human genome [ 27, 28], genetic screening is being proposed as a major opportunity for translating genetic and genomic advances into population health gains [ 29, 30]. Next generation sequencing (NGS) has already started to transform genetics, and clinical application of whole sequencing genetics has already shown to decrease the diagnostic odyssey in both adults and children [ 31, 32] and shortening intensive care units stay in critically ill children [ 33]. With decreasing costs and novel genotyping and sequencing technological platforms, interest in the use of molecular testing for NBS is increasing. However, DNA testing has already been introduced in NBS for several years for some disorders. As an example, most programs performing NBS for cystic fibrosis have implemented a CFTRmutation panel as a second-tier test following increased immunoreactive trypsin level. Considering the capacity to perform DNA testing from filter paper combined with the decreasing costs of DNA testing, there is growing interest for including more widely DNA testing technologies to NBS, which would greatly increase the capacity to identify rare but devastating disorders, but which also raises some ethical concerns. Table 2Main genes involved in core panel of diseases to be detected at birth according to ACMG newborn screening expert group (HGNC, HUGO Gene Nomenclature committee). Table 2Main genes involved in core panel of diseases to be detected at birth according to ACMG newborn screening expert group (HGNC, HUGO Gene Nomenclature committee). Group of conditions Condition HGNC approved gene symbol (OMIM)Organic acidemiasIsovaleric acidemia IVD (607036)Glutaric acidemia type I GCDH (608801)3-OH 3-CH3 glutaric aciduria HMGCL (613898)Holocarboxylase synthase deficiency HLCS (609018)Methylmalonic acidemia (mutase deficency) MMUT (609058)3-Methylcrotonyl-CoA carboxylase deficiency MCCC1 (609010), MCCC2 (609014)Methylmalonic acidemia (cobalamin disorders) MMAA (607481), MMAB (607568), MMADHC (611935)Propionic acidemia PCCA (232000), PCCB (232050)β-Ketothiolase deficiency ACAT1 (607809)Biotinidase deficiency BTD (609019)Disorders of fatty acid metabolismMedium-chain acyl-CoA dehydrogenase deficiency ACADM (607008 )Very long-chain acyl-CoA dehydrogenase deficiency ACADVL ( 609575)Long-chain L-3-OH acyl-CoA dehydrogenase deficiency HADHA (600890)Trifunctional protein deficiency HADHB (143450)Carnitine uptake defect SLC22A5 (603377)Carnitine transport defect CPT1A (600528), SLC25A20 (613698), CPT2 (600650)Disorders of amino acid metabolismPhenylketonuria PAH (612349)Maple syrup (urine) disease BCKDHA (608348), BCKDHB (248611), DBT (248610), PPM1K (611065)Homocystinuria (due to CBS deficiency) CBS (613381)Citrullinemia ASS1 (603470)Arginosuccinic acidemia ASL (608310)Tyrosinemia type I FAH (613871)HemoglobinopathiesSickle cell anemia (Hb SS disease) HBB (141900)Hb S/β-thalassemiaHb S/C diseaseEndocrinopathiesCongenital hypothyroidism DUOX2 (606759), DUOXA2 (612772), PAX8 (167415), TG (188450), TPO (606765), TSHR (603372), IYD (612025)…Congenital adrenal hyperplasia (21-hydroxylase deficiency) CYP21A2 (613815)Others metabolic conditionsGlycogen storage disease type II (Pompe) GAA (606800)Mucopolysaccharidosis type 1 IDUA (252800)X-linked adrenoleukodystrophy ABCD1 (300371)Classical galactosemia GALT (606999)OthersSpinal muscular atrophy due to homozygous deletion of exon 7 in SMN1 SMN1 (600354)Newborn hearing screening /Cystic fibosis CFTR (602421)Critical congenital heart disease /Severe combined immunodeficiencies ADA (608958), CARD11 (607210), CD247 (186780), CD3D (186790), CD3E (186830), CORO1A (605000), CTPS1 (123860), DCLRE1C (605988), IKBKB (603258), IL2RG (308380), IL7R (146661), JAK3 (600173), LAT (602354), LCK (153390), PRKDC (600899), PTPRC (151460), RAG1 (179615), RAG2 (179616) In a recent report, Yang et al.performed systematic NGS in biochemically screened-positive neonates by MSMS from three areas of the Jiangsu Province performing NBS for 27 inborn errors of metabolism (IEM). Over a period of four years, 536,008 neonates had positive 1 sttier tandem MS screen followed by MSMS retesting and targeted sequencing of 306 IEM-related genes from an extended panel of inherited metabolic diseases. A total of 1033 neonates were tested by NGS, followed by validation analyses by Sanger sequencing of positive findings. They identified the disease-causing genes of 194 cases for an overall incidence of 1:2763, which was higher than previously reported and suggesting that NGS allowed to identify additional cases [ 36]. Aminoacidopathies accounted for 43.5% of case findings, while 34.8% were organic acidemias and 21.7% were fatty acid oxidation disorders. Most cases needed clinical intervention and regular follow up, while only a few cases appeared to be asymptomatic at the time of publication. Long term follow up should allow to adjust conditions and genes to be included as part of the gene panel in routine NBS. The high throughput of NGS technology, which can carry out parallel sequencing of millions of DNA molecules, has the potential to increase the number of congenital disorders to be screened, in fact to a much higher order of magnitude seen with the introduction of tandem MS. In this context, methods for the selection of appropriate conditions to be introduced as part of a NGS NBS-panel must be developed and tested. With this in mind, Milko et al.recently developed an aged-based semi-quantitative scoring method for evaluating the relevance for the introduction of new genetic conditions to NBS panel using genomic sequencing [ 37]. Their model was mostly based on major screening criteria to evaluate clinical actionability (severity of the disease, likelihood of clinical manifestation, acceptability and efficacy of intervention, overall knowledge of the gene-disease association) [ 38]. This allowed them to identify 822 gene-disease pairs, with 466 classified as being children-onset with high clinical actionability, while another 245 gene-disease pairs were considered as low to no childhood actionability. Of note, validation of their method to reliably identify pediatric clinical actionability was performed by comparing how the 1 ryand 2 ryconditions of the RUSP [ 39] and conditions curated by the BabySeq project [ 40] performed with their approach. As expected, the correlations were not perfect, but they reflected the lower clinical actionability of 2 rycompared to 1 ryRUSP conditions, more of the latter being classified in the high clinical actionability category. Comparisons with the BabySeq projects, which categorized their gene-disease pairs on the validity of the gene-disease association, earliest reported age of onset, and penetrance, allowed to reach to a consensus for the identification of 292 conditions to be potentially introduced to a NGS NBS-panel, and another 125 optional for disclosure [ 37]. Considering that the benefit of NBS for many of the 466 disease-pairs is yet to be proven, it would be wise to apply a stringent classification restricting NGS NBS only for disorders showing definitive or strong clinical validity based on the ClinGen framework [ 41]. Of note, such genomic-based approaches may allow to help decision process about the debatable relevance of conditions such as 3-methylcrotonylglycinuria that are already part of the NBS panel of many jurisdictions. The gap between what is technologically possible by NGS and the capacity to offer appropriate services by public health systems is creating pressure to expand screening programs, often before adequate regulatory frameworks are in place [ 42, 43, 44]. Indeed, concerns are mainly related to psychological and physical consequences of broad genetic testing in asymptomatic children, such as parental anxiety in the presence of indeterminate findings, loss of confidentiality, and social stigma [ 25, 45, 46, 47, 48]. This is thus the subject of intense debates, especially in the absence of an appropriate medical intervention to offer. The generation of genomic information, not all of which is understood, potentially leads to significant risks associated with the release of incidental findings and variant of unknown significance (VUS). This new paradigm generated by the introduction of NGS in NBS must be taken into account by an appropriate public health framework, as recommended by Friedman et al. [ 44]. While some are already proposing that NBS programs incorporate, or even replace their current approach with genomic sequencing [ 49, 50], evidence-based methods should be implemented to inform the decision-making process and consider parameters such as prevalence and penetrance of the condition, false-positive findings, clinical outcome following the interventions, psychological impacts on families, and costs. In the case of NBS, the focus should be the best interest of the child, and thus the identification of conditions where timely interventions or prevention would prevent significant health harms to the child, not on genetic information that may potentially identify later-onset disorders which might need medical attention. Otherwise there would be a denial of the autonomy of the individual. The introduction of NGS in NBS carries an immense potential to better identify children with genetic disorders and improve outcomes, but also poses important ethical issues. It is not surprising that it is the subject of active discussions and debates. 3. NBS in Low-Resource Areas: An Overview Even if NBS is a considered as one of the major public health realization [ 51], unfortunately many countries in low resource-areas do not have a structured national program. While the incidence of congenital disorders is estimated at 8 million of annual birth worldwide, only one-third of neonates are receiving NBS for at least one inborn disorder [ 52]. In addition to the lack of funding and of specialized human resources, NBS advocates face many barriers to NBS implementations. Also, the centralized model of NBS based on the shipment of collection papers to a central NBS laboratory may not be suitable in these areas. In the absence of a structured public health system that can insure effective care and follow up of screen-positive neonates, NBS may not be viable. Indeed, NBS includes a whole series of activities and a sequence of actions, it is not only a testing laboratory. Blood collection and transport to a central laboratory, follow up investigations and treatments of those with an abnormal result requires a structured public health system and adequate infrastructure. In addition, the different realities of outlying and urban areas make it difficult to implement a uniform NBS program. This was emphasized in India where it was suggested that NBS for congenital hypothyroidism only be introduced in the countryside, while screening should be implemented for congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH) and G6PD deficiency in urban areas, tandem mass spectroscopy being reserved only for investigating the high-risk cases. This must hold true for most of the developing countries [ 53]. In addition, health care authorities may not see NBS of rare conditions, sickle cell diseases aside for some territories, as a priority. This will possibly be attenuated as more neonatal and children’s suffering from more common disorders are being taken care of. Apparently, screening for genetic disorders and birth defects becomes relevant when infant mortality rate falls below 40 per 1000 live births [ 54], and become a public health priority when national infant mortality rate drops into single digits, i.e. below 10 per 1000 live births [ 54, 55]. However, differences in the population genetic background will inevitably lead to differences in the NBS panel. Indeed, if NBS for CH and some inborn errors of metabolism were found to be cost-effective in countries from the northern hemisphere, screening for disorders as hemoglobin disorders and glucose-6 phosphate dehydrogenase deficiency may eventually prove to be cost-effective in sub-Saharian Africa, India and South-East Asia where these disorders are prevalent. Finally, as proposed by Therrell and Padilla, there should be increased emphasis on point-of care (POC), taking advantage of newborn hearing screening and screening for critical congenital heart defects performed at the bedside [ 56]. Clear benefits should be achieved by the integration of these NBS modalities into a more comprehensive NBS infrastructure. Pilot projects should continue to be implemented in low-resource areas using a strategy that is integrated with current clinical care, possibly starting with hemoglobin disorders, with specialized expertise centered around one genetic service, and with the objective to evaluate the reduction of the genetic burden, cost-benefits, and barriers to implementation to help provide efficient NBS. However, it is unfortunate that the latest WHO recommendations on newborn health guidelines published in May 2017 did not identify NBS in the promotion of newborn health [ 57]. Progresses in the implementation of NBS in developing countries worldwide was thoroughly reviewed recently [ 56]. 4. Conclusion and Perspectives NBS is considered as a major public health success of the 20 thcentury. NBS performed during the 1 stfew days of life through blood spot sampling on a filter paper followed by blood testing can prevent morbidity and mortality from severe conditions. NBS programs evolved from screening for one or a few disorders to potentially more than 50 conditions, thanks to the enhanced capacity of mass spectrometry to identify simultaneously multiple disorders with high sensitivity from only one sample injection into the instrument. However, there is still significant heterogeneity in the NBS panel offered by the different jurisdictions, in part due to variations in population genetics, but also in the various approaches and interpretation of the WHO screening criteria and decision-making process in the face of scarce data on disorders that are individually rare. Following the wave of large-scale NBS expansion around the 2000’s in many jurisdictions triggered by the introduction of MSMS, the implementation of high throughput NGS, which has already started to transform clinical genetics, has the potential to bring the NBS area to another level. Used on a large-scale, whole genome sequencing will identify another set of rare neonatal but devastating disorders for which presymptomatic interventions are available. It also has the potential to prevent misdiagnosis, shorten delay in diagnosis, facilitate early intervention, and improve diagnosis. However, there is a scarcity of data on most IEM, which may have highly variable clinical manifestations. The incorporation of NGS technology in NBS panels will need a stringent decision process that will consider evidence-based reliable methods from large-scale studies. Importantly, ethical concerns related to the generation of genomic information, a large proportion of which we still don’t understand the clinical significance, will need to be addressed. As “too much screening is bad screening”, the balance between benefit and harm must still be taken into consideration when evaluating the capacity of NGS to improve child’s care. It should not be damped by precipitous, lax, and potentially harmful decision processes. Finally, while about two-thirds of infants worldwide are born in limited-resource areas that still do not have any NBS infrastructure, several initiatives worldwide show promises, but are also faced with many barriers of implementation and sustainability. It would be a pity to come to the realization that the northern hemisphere continues to apply technological advances for the benefit of neonates and children, and the population at large, while low-resource areas are still struggling to implement a basic NBS infrastructure. Author Contributions YG designed the manuscript plan. YG and DB wrote the initial version of the manuscript and agree with its final version. Competing Interests The authors declare that they have no competing interests. References Guthrie R. Blood screening for phenylketonuria. JAMA. 1961; 178: 863. [ CrossRef ] Guthrie R, Susi A. 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CAS:146-91-8 - FACTA Search Query: CAS:146-91-8 ( GDP ) 11,213 Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for Gsalpha. A novel Gsalpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (Gsalpha R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, Gsalpha expression in erythrocyte membranes from the affected patient was reduced by 50%. A Gsalpha R258W cDNA, as well as one with arginine replaced by alanine (Gsalpha R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine- 5'-O-(3-thiotriphosphate) (GTPgammaS) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 microM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPgammaS or AlF4- was assessed by trypsin protection assays. Both mutants were protected normally by GTPgammaS but showed reduced protection in the presence of AlF4-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP . A Gsalpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type Gsalpha. Sucrose density gradient analysis demonstrated that both Gsalpha R258W and Gsalpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPgammaS or 2 mM GDP . The crystal structure of Gsalpha demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP . ... PMID:A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation. 972 13 We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct. ... PMID:Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation. 998 42 Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Galpha12/13. Galpha13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Galpha13 using an assay for G protein alpha-chain activation that is based on the fact that an activated (GTP-bound) alpha-chain is resistant to trypsinization compared with an inactive ( GDP -bound) alpha-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Galpha13 from degradation by trypsin . In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and alpha13, ET-3 and 5'-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of alpha13 compared with Gpp(NH)p alone. The specificity of ETB receptor-alpha13 coupling was documented by showing that beta2 receptors and isoproterenol or ETA receptors and ET-1 did not activate alpha13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of alpha13. ... PMID:Endothelin-B receptors activate Galpha13. 1019 25 The chicken pineal gland is a photosensitive neuroendocrine organ producing melatonin in circadian clock-regulated and light-sensitive manners. To understand the relationship between the photoreceptive molecule pinopsin and the light-dependent melatonin suppression that is sensitive to pertussis toxin treatment, we have searched for pertussis toxin-sensitive G protein alpha-subunits expressed in the chicken pineal gland. Here we report the cDNA cloning of the pineal transducin alpha-subunit (Gtalpha), which is highly homologous to human retinal rod cell-specific Gt(1)alpha. Concurrent cDNA cloning of chicken retinal Gt(1)alpha and Gt(2)alpha (rod and cone cell-specific alpha-subunits of transducin, respectively) revealed that the chicken pineal Gtalpha is identical to the retinal Gt(1)alpha. Double-immunostaining analysis of the chicken pineal sections localized Gt(1)alpha-immunoreactivity in the rudimentary outer segments of both follicular and parafollicular pinealocytes that were immunopositive to anti-pinopsin antibody. To examine whether pineal Gt(1)alpha is involved in the pineal phototransduction pathway, trypsin protection assay was applied for detecting the conversion of GDP -bound Gt(1)alpha into the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound form in the pineal membrane homogenate. It was clearly demonstrated that the pineal Gt(1)alpha is activated in a light-dependent manner in the presence of GTPgammaS. These data together suggest strongly that pineal Gt(1)alpha mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes. ... PMID:Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland. 1085 64 When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)- GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors. ... PMID:G protein beta 5 subunit interactions with alpha subunits and effectors. 1098 79 Among the prokaryotae, the nucleotide ppGpp is a second messenger of physiological stress and starvation. The target of ppGpp is RNA polymerase, where it putatively binds and alters the enzyme's activity. Previous data had implicated the beta-subunit of Escherichia coli RNA polymerase as containing a single ppGpp binding site. In this study, a photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp), was used to localize the ppGpp binding site. In in vitro transcription assays, 6-thio-ppGpp inhibited transcription from the argT promoter identically to bona fide ppGpp. The thio group of 6-thio-ppGpp is directly photoactivatable and is thus a zero-length cross-linker. Cross-linking of RNA polymerase was directed primarily to the beta'-subunit and could be competed efficiently by native ppGpp but not by GTP or GDP . Cyanogen bromide digestion analysis of the cross-linked beta'-subunit was consistent with an extreme N-terminal cross-link. To assess allosteric consequences of ppGpp binding to RNA polymerase, high level trypsin resistance in the presence and absence of ppGpp was monitored. Trypsin digestion of RNA polymerase bound to ppGpp leads to protection of an N-terminal fragment of the beta'-subunit and a C-terminal fragment of the beta-subunit. We propose that the N terminus of beta' together with the C terminus of beta constitute a modular ppGpp binding site. ... PMID:Binding of the transcription effector ppGpp to Escherichia coli RNA polymerase is allosteric, modular, and occurs near the N terminus of the beta'-subunit. 1103 17 G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin -resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface. ... PMID:S111N mutation in the helical domain of human Gs(alpha) reduces its GDP/GTP exchange rate. 1196 1 G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the beta subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gbetagamma dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gbeta-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G(i/o) proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gbetagamma dimers. In the retinal NDPK B-enriched fractions, a Gbeta-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gbetagamma. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gbetagamma. For identification of the phosphorylated histidine residue, bovine brain Gbetagamma and G(t)betagamma were thiophosphorylated with guanosine 5'-O-(3-[(35)S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin , separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine Gbeta(1) that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with Gbetagamma dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gbeta(1) subunits. ... PMID:Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Complex formation of NDPK B with Gbeta gamma dimers and phosphorylation of His-266 IN Gbeta. 1248 23 Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP -bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP -bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states. ... PMID:A peptide core motif for binding to heterotrimeric G protein alpha subunits. 1605 11 The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-alpha (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of alphaT to undergo rhodopsin-stimulated GDP -GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP -bound forms of alphaT, alphaT(R238A) and alphaT(R238K), the alphaT(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT(R238E) effectively blocked rhodopsin-catalyzed GDP -GTP exchange on alphaT, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT(R238E) exhibited a resistance to trypsin -proteolysis similar to that observed with activated forms of alphaT. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative G alpha subunit that can effectively block signaling by G protein-coupled receptors. ... PMID:A switch 3 point mutation in the alpha subunit of transducin yields a unique dominant-negative inhibitor. 1610 22	http://www.nactem.ac.uk/facta/cgi-bin/facta3.cgi?query=CAS%3A146-91-8%7C111111%7C0%7C0%7C236100%7C90%7C100
Medicines \| Free Full-Text \| The Medial Surface of the Auricle: Historical and Recent Maps. What Are the Possible Expectations of the & ldquo;Thumb-Index Technique& rdquo; Introduction: The medial surface of the auricle (MSotA), as compared to the lateral, has been less studied and has limited consensus among schools of auricular acupuncture (AA) due to its small size, greater difficulty in carrying out an adequate physical examination on it, and less precise and limited agreement on its anatomical references as compared to the lateral surface. The thumb-index technique TIT is performed using a guiding finger (taking advantage of the anatomical conformation of the lateral surface) to guide the explorer finger (placed on the MSotA) to project the therapeutic areas and land marks on the MSotA. TIT could be considered useful and effective in AA to make the most of diagnostic and therapeutic MSotA potential. Methods: An investigation was carried out on the impact of TIT in AA practice through a survey collected from former AA students. Results: TIT showed a high consensus, and is used and appreciated by AA practitioners. Discussion/ Conclusions: To date, in AA, there is no thoroughly shared nomenclature for MSotA. TIT is simple and quick to project on to MSotA the well-coded lateral surface auricular maps from French or Chinese AA schools. The Medial Surface of the Auricle: Historical and Recent Maps. What Are the Possible Expectations of the “Thumb-Index Technique” by Antonello Lovato 1,2,* , Francesco Ceccherelli 2,3 , Giuseppe Gagliardi 2,4 and Marco Postiglione 2,5 1 A.I.R.A.S, Italian Association for the Research and the Scientific Update, 35100 Padua, Italy 3 Anesthesiology and Pain Therapy, Department of Anesthesiology and Reanimation, University of Padua, 35100 Padova, Italy 4 Postgraduate Specialization in Anesthesiology Specialist, Postgraduate Specialization in Sport Medicine Specialty, ULSS 5 Polesana, 45100 Rovigo, Italy 5 Postgraduate Specialization in Public Health and Preventive Medicine, Postgraduate Specialization in Physical and Rehabilitation Medicine, ULSS 5 Polesana, 45100 Rovigo, Italy * Author to whom correspondence should be addressed. Medicines 2022 , 9 (2), 13; https://doi.org/10.3390/medicines9020013 Received: 13 November 2021 / Revised: 31 January 2022 / Accepted: 14 February 2022 / Published: 17 February 2022 (This article belongs to the Special Issue Medicines: Identifying, Exposing and Tackling Unethical and Corrupt Practices is Essential for Safeguarding Patient and Public Health Interests Worldwide ) Abstract : Introduction: The medial surface of the auricle (MSotA), as compared to the lateral, has been less studied and has limited consensus among schools of auricular acupuncture (AA) due to its small size, greater difficulty in carrying out an adequate physical examination on it, and less precise and limited agreement on its anatomical references as compared to the lateral surface. The thumb-index technique TIT is performed using a guiding finger (taking advantage of the anatomical conformation of the lateral surface) to guide the explorer finger (placed on the MSotA) to project the therapeutic areas and land marks on the MSotA. TIT could be considered useful and effective in AA to make the most of diagnostic and therapeutic MSotA potential. Methods: An investigation was carried out on the impact of TIT in AA practice through a survey collected from former AA students. Results: TIT showed a high consensus, and is used and appreciated by AA practitioners. Discussion/ Conclusions: To date, in AA, there is no thoroughly shared nomenclature for MSotA. TIT is simple and quick to project on to MSotA the well-coded lateral surface auricular maps from French or Chinese AA schools. Keywords: auriculotherapy ; ; 1. Introduction: 1.1. The Medial Surface of the Auricle (or MSotA) The medial surface of the auricle (or MSotA), as compared to the lateral surface, has been less studied and has limited consensus among the different schools of auricular acupuncture (AA). This is due to the small size, the greater difficulty in carrying out an adequate physical examination on it, and the less precise and limited agreement on its anatomical references as compared to the lateral surface. There has been much discussion on the innervation of the MSotA. From a study conducted on the innervation of the auricle of human cadavers, the medial surface can be divided into three parts [ 1 ]. The upper third of the MSotA is innervated by ATN (auriculo-temporal nerve of the trigeminal nerve) and GAN (great auricular nerve from the cervical plexus C2-3), the middle third is innervated mainly by ABVN (auricular branch of vagus nerve) and partially by GAN, and the lower third is innervated mainly by GAN and to some degree by ABVN, respectively. In November 1990, WHO organized a working group on auricular nomenclature in Lyone (France). In this meeting, it was established that the MSotA can be divided into four parts as follows: posterior peripheral (PP), posterior intermediate (PI), posterior central (PC), and posterior lobular (PL), respectively [ 2 ]. To date, there is no fully shared nomenclature of the MSotA despite the efforts made by the Word Federation of Chinese Medicine Societies (WFCMS) during the last meeting held in Lyon in June 2012 [ 3 ]. 1.2. Historical Maps In China, great importance has always been given to AA diagnosis based on the inspection of the auricle. AA in China has never been systematically defined with the use of maps and has never had its own development (unlike somatic acupuncture), although it was performed earlier than in France. The auricle was mentioned in ancient Chinese medicine texts, such as the Yin–Yang Eleven Channel Moxibustion ( Yinyang Shiyi Mai Jiujing ), written during the Warring States period and Spring and Autumn Period, which is between 770 and 221 BC [ 4 ]. A first description, though very primitive, of the auricle appears in the Linh Shu (474-221 BC). In a book written during the Ming Dynasty (1368AD-1644AD) by Zhou Yufan called Massage Technique for Children ( Xiaoer Anmo Shu ), for the first time, the five Zang organs (i.e., heart, liver, spleen, lung, and kidney) theory was described as regards to their correspondence to the MSotA [ 4 ]. Based on this theory, Zhenjun Zang produced the first AA map in a book published in 1888 Essential Techniques for Massage ( Lizheng Anmo Yaosu ) [ 5 ]. 1.3. Recent Maps Starting from clinical observations, in 1957, P. Nogier proposed the somatotopy of the auricle, creating a map that has become a reference for all practitioners from East to West. The map was drawn by G. Bachmann, based on the proposal of P. Nogier, and published in a German magazine Deutsche Zeitschrift für Akupunktur [ 6 , 7 ]. This map was used as the gold standard for AA, although there is no mention of the MSotA as shown in Figure 1 and Figure 2 . This significant idea of P. Nogier traveled the world and arrived in China, where, based on Nogier’s work, a very simple first map was drawn, which was probably reserved for military doctors. Chinese doctors learned to use AA as a “microsystem” and, as time passed and experience increased, more auricular points were discovered. In 1971, H. Jerricot, an illustrious French physician, cited the Chinese map of the auricle, which still only described the lateral surface [ 8 ], as shown in Figure 3 . In 1975, given the lack of information on the MSotA in the first 1957 auricle map, Nogier, together with Boudiol and Bahr, published a map representing the musculoskeletal system and visceral areas on the MSotA [ 9 ]. In 1981, Marco Romoli proposed and published his first septogram. In this first septogram, Romoli also drew the medial surface. Strictly speaking, the septogram cannot be defined as a map but is rather a tool used to delineate the skin changes detected at the auricle on a two-dimensional image to formulate an accurate AA diagnosis [ 10 ]. In 1988, the Chinese Association of Acupuncture and Moxibustion (CAAM) published an article called “The project of the standardization of auricular acupuncture points”, in which standardization of the lateral and medial surface of the auricle was proposed. The lateral surface was divided into areas that largely reflected the anatomy of the lateral surface of the auricle, integrated by AA points placed in areas that were not visible on the map. The medial surface, on the other hand, recalled the Chinese historical map, which was connected with the five Zang organs. This map is still recognized as the Nanjing Map and represents a reference point for Chinese standardization [ 11 ]. In 1990, Terry Oleson proposed his second map, which was considered innovative in many ways. It highlighted the hidden parts of the auricle on both the lateral and medial surfaces. Furthermore, the mapping of auricle areas by Oleson was characterized by being very adaptable to any auricle because it had no fixed areas, instead showing these areas as being proportionally divided into the individual parts of the auricle similar to the “CUN” measurement used in somatic acupuncture. Oleson’s map (as Romoli’s septogram) cannot be properly defined as a map as the author did not propose his own points but delineated the position of points from the French and Chinese schools. The map essentially considered (with one exception on the lateral surface and three on the medial surface) the nomenclature of the areas of the auricle established by WHO in 1990 in Lyon (France) [ 12 ]. Oleson recognized and described 24 auricular landmarks (LMs) on the lateral surface, which are identified as well-defined areas used as landmarks for researching specific therapeutic auricle zones. LMs are not necessarily therapeutic zones and, in a study carried out by Oleson himself, they were found to be reliable reference points since the distances between the various LMs were found to be fairly constant with a relatively low standard deviation, regardless of race, age, and sex [ 13 ]. This map is shown in Figure 4 . In 2003, Marco Romoli published his book on AA Agopuntura Auricolare (published by UTET in 2003), in which the septogram was modified and the image of the MSotA was dropped [ 14 ]. Furthermore, it became “flexible” since the rays of the septogram were not fixed but created by drawing three lines that connected specific anatomical references to the point zero, thus dividing the auricle into three macro-sectors. Then, each macro-sector was divided into a specific number of smaller sectors by lines drawn from the point zero, which were proportional to the three macro-sectors previously delineated, as shown in Figure 5 . In 2010, David Alimi presented a new nomenclature and AA cartography to WHO through the World Federation of Chinese Medicine Societies (WFCMS). This AA map was centered on the prime zero point (or corpus callosum point) rather than on the point zero, unlike Romoli’s septogram. This map marked the points on the lateral surface in a different way according to their localization and a different color was used based on the embryological origin of the tissue represented therein. On this map, the AA points on the MSotA were all drawn with the same symbol (i.e., a square) to mark the difference with points found on the lateral surface of the auricle. The coloring of AA points represented the embryological origin of the tissue represented therein. This map consisted of 20 rays all with the same angle. The limit was that this map was not as “flexible” as others since it had to be customized to each patient and it did not highlight hidden areas as well as Oleson’s map [ 15 , 16 ]. This map is shown in Figure 6 . A recent publication by Lei Wang et al. aimed to verify the correspondence between AA points as proposed by the Chinese system (as represented by the World Federation of Acupuncture-Moxibustion Societies or WFAS) and as proposed by the European system (i.e., according to Nogier and Bahr) [ 17 ]. The authors concluded that 24 AA points have the same name and position on the auricle. Furthermore, 25 areas or points maintain the same nomenclature but represent body parts that are different between the 2 systems and therefore have different therapeutic indications. In 2016, Pei-Jing Rong analyzed 9 methods to localize AA points, with the aim of highlighting the strengths and disadvantages of each of these methods [ 18 ]. The paper concluded that the international method proposed by the WFAS based on lines and points was an appropriate method. The same paper recognized that the areas described and proposed by the WFAS for the MSotA were too large and that an effort to improve the localization of the AA points of the MSotA was desirable. Andres Wirz-Ridolfi explained that for AA to be effective, it must be very precise [ 4 ]. This concept has been underlined by works carried out with functional magnetic resonance imaging (fRMN) in recent studies, such as those by David Alimi in 2002 and 2014 [ 19 , 20 ] and by Marco Romoli in 2015 [ 21 ]. The author stressed the need to have better and more comparable maps to obtain greater international consensus on AA points and greater international credibility. Furthermore, the author stated that scientific recognition is certainly important but the final goal (to be considered even more important) is to obtain good therapeutic results, which is possible only if the correct AA points of the auricle are treated (i.e., punctured by the acupuncture needles or irradiated by LASER). 1.4. Critical Points to Mapping the MSotA The MSotA could be described as an undefined area as it is the least studied of the auricle. The anatomical structure, small size (as compared to the lateral surface), and fewer anatomical references contribute to making MSotA be the least studied and known area of the auricle. The map Oleson published in 1990 helped to highlight the hidden areas of the medial surface. Oleson, in his most recent map ( Figure 7 ), divided the medial surface into five parts: posterior periphery (PP), posterior concha (PC), posterior triangle (PT), posterior groove (PG), and posterior lobe (PL), respectively. Therefore, there is no shared nomenclature of the MSotA to date [ 12 ]. Another limit of Oleson’s map is that it shows the MSotA only as “open” and therefore it does not correlate with the real image of the patient. Similarly to the lateral surface, it would be desirable to have a natural and “open” image. If the concept of a three-dimensional representation of the organs and anatomical structures on the auricle is achieved, considering the lateral and medial surfaces with completely different maps appears difficult. On the other hand, drawing two maps, one for the lateral surface and one for the MSotA, means considering that the two surfaces of the auricle are completely disconnected from each other. Furthermore, it means that the therapeutic area has a superficial and two-dimensional value ( Figure 8 ). 1.5. The Thumb-Index Technique To overcome these difficulties and to make the most of the diagnostic and therapeutic potential of MSotA, the authors propose a method that can be used in everyday AA practice. The method aims to project the points and areas of the auricle and, where possible, the LMs (auricular landmarks) described by Oleson of the lateral surface on the MSotA. The method proposed is the thumb-index technique (or TIT). This method uses a guiding finger and an explorer finger. The guiding finger takes advantage of the anatomical conformation of the lateral surface to guide the explorer finger placed on the MSotA. This allows the practitioner to project the therapeutic areas and the LMs on the MSotA. The result is a narrower or flattened projection of the homunculus on the MSotA while the practitioner can never lose the overall understanding of the area explored and, above all, avoids two distinct and discordant results when examining the lateral surface and the MSotA. This simple and quick technique also allows the points of the lateral surface identified by both the French and Chinese Schools of AA to be projected on the MSotA. The TIT is shown in Figure 9 . The purpose of this article is to understand the new, quick, and simple thumb-index technique and whether it could be considered useful and effective to make the most of the diagnostic and therapeutic potential of the medial surface of the auricle in auricular acupuncture. 2. Materials and Method An investigation was carried out on the impact of TIT in AA practice. The investigation was carried out by administering a set of questions on TIT to medical doctors who practice AA. The subjects selected were medical doctors, who were former AA course students of the past 5 years (from 2016 to 2020) who obtained the AA practitioner diploma from the AIRAS AA School in Padova (Italy). A request to participate in the study was sent via email together with the set of 7 questions to answer. The set of 7 questions were formulated as follows: (1) How many years have you been practicing auricular acupuncture (AA)? (2) Do you use the MSotA when practicing AA? (3) On a scale from 1 to 10 (where 1 is easy and 10 is very difficult), how difficult is it for you to locate therapeutic areas on the MSotA? (4) On a scale from 1 to 10 (where 1 is not important and 10 is very important), how important would it be to have a quick and simple method to be able to locate therapeutic areas on the MSotA? (5) Do you know the thumb-index technique (or TIT)? After a brief explanation of the TIT, the last two questions were asked. (6) On a scale from 1 to 10 (where 1 is not much and 10 is a lot), how much do you think TIT would help locate therapeutic areas on the MSotA? (7) On a scale from 1 to 10 (where 1 is never and 10 is always), now that you know this method would you include it in your everyday AA practice? The answers were recorded and evaluated. The gender and age of the subjects who answered the survey were recorded. 3. Results A total of 108 subjects were contacted and asked to answer the set of questions. A total of 70 subjects completed the survey, of which 58.6% were male and 41.4% were female with a mean age 51.94 years (SD ± 12.07, maximum and minimum age of 70 and 32 years, respectively). Table 1 includes the results of the survey sent to the investigated subjects. The results listed include the mean score, standard deviation (SD), mode, and maximum and minimum score. Questions are listed in the materials and methods by number. The subjects declared had been practicing auricular acupuncture for a mean of 4.81 years (SD 4.92, maximum and minimum score of 20 and 1, respectively). Of the subjects interviewed, 80% admitted to using the MSotA in their everyday AA practice. More than 80% of subjects agreed on the importance of having a quick and simple method to be able to locate therapeutic areas on the MSotA. As many as 81% stated that they had knowledge of TIT. Nearly all subjects agreed, when answering the last question of the survey, that they would include TIT in their everyday AA practice now that they knew the method, with a mean score of 8.25 (SD 1.17, maximum and minimum score of 10 and 5, respectively) on a scale from 1 to 10 (where 1 is never and 10 is always). 4. Discussion and Conclusions To date, there has been no nomenclature of the MSotA areas that is thoroughly shared by the various AA schools. Oleson’s map has the advantage of being flexible as it adapts to the auricle regardless of its shape and size. Oleson described 24 LMs on the lateral surface, which are reference points used for searching for further therapeutic or diagnostic areas. However, LMs are not described for the MSotA; thus they must eventually be deduced by the AA practitioner. The acupuncture point has a three-dimensional value, being primarily located at sites that correspond to the neurovascular bundles of blood plexuses and myelinated and unmyelinated nervous fibers. It is reasonable to believe that drawing the acupuncture point on a map of the lateral surface of the auricle and, again, on a map on the MSotA downgrades its value from its three-dimensional concept to a two-dimensional interpretation. To circumvent these critical points and avoid tedious and time-consuming practices, the practitioner can easily find the corresponding reference points of the lateral surface of the auricle and project them on the MSotA using the TIT. TIT is considered to be a well-accepted method since it is a simple and quick way to project the well-coded auricular maps of the lateral surface on the MSotA, regardless of whether they are from French or Chinese AA schools. Furthermore, this method also considers the three-dimensional structure of the acupuncture point and the likely three-dimensional representation of organs and systems on the auricle surface. It is reasonable to believe that the mapping the MSotA for further somatotopies is a difficult and inefficient exercise that can easily be overcome with the thumb-index technique. Author Contributions Conceptualization, A.L.; Methodology, F.C.; Formal Analysis, G.G.; Writing, review and editing, M.P. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. References Peuker, E.T.; Filler, T.J. The nerve supply of the human auricle. Clin. Anat. 2002 , 15 , 35–37. [ Google Scholar ] [ CrossRef ] [ PubMed ] WHO. 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Legend of Figure 2 : 1. Bladder; 2. Kidney; 3. Pancreas; 3a. Gallbladder; 4. Liver; 5a. Esophagus; 5b. Cardia; 5c. Stomach; 5d. Small Intestine; 5e. Large Intestine; 6. Lung; 7. Heart; 8. Subcortex; 9. Internal Nose; 10. Endocrine; 11. Spleen. Figure 3. Chinese map cited by MD Henri Jarricot, 1971. Figure 3. Chinese map cited by MD Henri Jarricot, 1971. Figure 4. Oleson’s map highlighting the hidden parts of the auricle, 1980. Figure 4. Oleson’s map highlighting the hidden parts of the auricle, 1980. Figure 5. Second version of Romoli’s septogram, 2003. Figure 5. Second version of Romoli’s septogram, 2003. Figure 6. The medial surface of the map by D. Alimì, 2010. Figure 6. The medial surface of the map by D. Alimì, 2010. Figure 7. Medial surface nomenclature WHO Lyon 1990. Legend: PP: Posterior Peripheral; PI: Posterior Intermediate; PC: Posterior Central; PL: Posterior Lobular. Figure 7. Medial surface nomenclature WHO Lyon 1990. Legend: PP: Posterior Peripheral; PI: Posterior Intermediate; PC: Posterior Central; PL: Posterior Lobular. Figure 8. Lateral and medial surface of the auricle by A. Lovato 2019, with the division into areas according to the nomenclature of T. Oleson. Figure 8. Lateral and medial surface of the auricle by A. Lovato 2019, with the division into areas according to the nomenclature of T. Oleson. Figure 9. The thumb-index technique (TIT). Figure 9. The thumb-index technique (TIT). Table 1. The results obtained from the survey are illustrated. Table 1. The results obtained from the survey are illustrated. Question Mean SD Max Score Min Score 1 4.81 ±4.92 20 1 3 6.14 ±2.17 10 1 4 8.25 ±2.25 10 1 5 0.81 ±0.39 1 0 6 8.17 ±1.85 10 1 7 8.25 ±1.17 10 1 © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). MDPI and ACS Style Lovato, A.; Ceccherelli, F.; Gagliardi, G.; Postiglione, M. The Medial Surface of the Auricle: Historical and Recent Maps. What Are the Possible Expectations of the “Thumb-Index Technique”. Medicines 2022, 9, 13. https://doi.org/10.3390/medicines9020013 AMA Style Lovato A, Ceccherelli F, Gagliardi G, Postiglione M. The Medial Surface of the Auricle: Historical and Recent Maps. What Are the Possible Expectations of the “Thumb-Index Technique”. Medicines. 2022; 9(2):13. https://doi.org/10.3390/medicines9020013 Chicago/Turabian Style Lovato, Antonello, Francesco Ceccherelli, Giuseppe Gagliardi, and Marco Postiglione. 2022. "The Medial Surface of the Auricle: Historical and Recent Maps. What Are the Possible Expectations of the “Thumb-Index Technique”" Medicines9, no. 2: 13. https://doi.org/10.3390/medicines9020013	https://www.mdpi.com/2305-6320/9/2/13
The Oxford Oratory A sanctuary in the midst of the city The Oxford Oratory is a vibrant centre of Catholic life. Our church is open every day: join us for Mass, pop in for some quiet prayer, or come and discover more at one of our groups. Our historic church of St Aloysius has been a key feature in the lives of the city’s Catholics for 150 years, attracting people of all ages and from every walk of life. We use beauty to raise hearts and minds to God, faithful to the traditions of St Philip Neri and St John Henry Newman. The Oratory Prayer Book is now shipping internationally! We’ve made it even easier to buy the Oratory Prayer Book from anywhere in the world. And we’ve made it easier to order multiple copies too — in the UK and worldwide. Order your copy here:https://tinyurl.com/opb-buy If you have one, we’d love to hear what you think about it. Let us know in the comments, or tag a photo of your prayer book in the wild with #oratoryprayerbook Thanks to @acatholicinoxford for the photo. #oxfordoratory Worship What does it actually mean to worship God? In the New Testament, the word we most often translate as ‘worship’ doesn’t mean to reverence another in some abstract sense. It always involves some kind of bodily action. When we think about the magi visiting the Holy Family, we sometimes convey this motion by describing them — after they fall to their knees — as ‘doing homage’. But it’s actually that same word here we might translate elsewhere as ‘worship’. It’s just that we recognise the need to describe the bodily sign of their submission and adoration of the new born King with something that sounds a bit more physical. The word we usually translate as ‘worship’ ( proskuneo) comes literally from a root meaning ‘to make yourself like a dog before the other person’. It originally meant prostrating yourself on the floor to show your humble submission before another. By the time of the New Testament, people wouldn’t necessarily have had the root of this word in mind whenever they used it,* but the word still carries with it the idea of doing something with your body to express your worship. The feast of Corpus Christi is all about this kind of bodily worship. It’s tied to the fact that we are not pure spirits, but that we are human beings, body and soul. To worship God is not just a spiritual act. The right worship of God does not consist in simply having the right kinds of thoughts towards him. But the worship of God involves the humble submission of our entire human being, body and soul, before the God who is greater than all of us. And ever since the Incarnation, we do this not just before an abstract spirit. Because, from the moment of the Incarnation, God really does have a human body, that we can worship, that we fall down on our knees before, like the magi and the Canaanite woman, and so many others in the Gospel who kneel down and worship Christ. When we worship God, we kneel before God who is present in the flesh, under the appearances of bread and wine, in our churches all over the world. Corpus Christi is a joyful celebration of Christ’s presence among us, and it gives us an opportunity to think about how we respond to that gift. All this talk of kneeling and worship might not sound so joyful, but one of the great paradoxes of Christianity is that we find our greatest freedom in the humble service of God. Our greatest joy really should be found in finding ways to express this loving worship. So at least once a year, it is no bad thing to think about how we express with our own bodies that worship of God’s body, and what ways we can find to worship him even more perfectly. This Sunday, we will carry Christ’s Body through the streets of our city. We have a chance to demonstrate to the world our love, our joyful service, our worship of him, through our presence. The procession leaves our church at 2:30pm. Make sure you are there. But we should also think about the more ordinary things, like making sure we show that worship whenever we are in his presence. We should show through our behaviour in church that we believe him to be there: by genuflecting before him in the tabernacle of the church, and by keeping the church quiet and prayerful. We show our worship above all by kneeling down before him — like all those people in the Gospel — for the moment of Holy Communion. And we allow the priest to place the Host on our tongues, so that we don’t handle unnecessarily what is holy and precious, and no particles of Our Lord’s Body are ever dropped or lost through carelessness. We continue that worship in our prayers of thanksgiving after Communion and once the Mass has ended. We certainly never leave church during Communion. And we might think of ways of extending the time we spend with Christ present in the Blessed Sacrament: by attending Mass during the week sometimes, or visiting him during the times each week when his Body is exposed solemnly on the altar for Adoration, or even just by visiting an empty church, where he is waiting for us to show him our worship. Our Lord is hidden there, waiting for us to come and visit him, and make our request to him. See how good he is! He is there in the Sacrament of his love, sighing and interceding incessantly with his Father for sinners. To what outrages does he not expose himself, that he may remain in the midst of us! He is there to console us; and therefore we ought often to visit him. How pleasing to him is the short quarter of an hour that we steal from our occupations, from something of no use, to come and pray to him, to visit him, to console him for all the outrages he receives! When he sees pure souls coming eagerly to him, he smiles upon them. They come with that simplicity which pleases him so much, to ask his pardon for all sinners, for the outrages of so many ungrateful men. What happiness do we not feel in the presence of God, when we find ourselves alone at his feet before the holy tabernacles! ‘Come, my soul, redouble thy fervour; thou art alone adoring thy God. His eyes rest upon thee alone.’ — St John Mary Vianney * One exception is that I do think Christ had this root meaning in mind when he spoke to the Canaanite woman in Matthew 15:21–28. He wasn’t randomly insulting her by comparing her to a dog, but drawing attention to the fact that she was making herself like a dog before him — i.e. worshipping him — because she recognised him to be God. In other words, he was acknowledging her worship as proof of her faith and humility. On Saturday, Fr Rupert led Men’s Oratory on a walking pilgrimage to Islip, birthplace of St Edward the Confessor. #oxfordoratory Congratulations to Elizabeth who was received into the Church yesterday evening on the Feast of the Visitation. #oxfordoratory Congratulations to Livi, baptised on the vigil of Pentecost. “Therefore, Lord, we pray: graciously accept this oblation of our service, that of your whole family, which we make to you also for those to whom you have been pleased to give the new birth of water and the Holy Spirit, granting them forgiveness of all their sins; order our days in your peace, and command that we be delivered from eternal damnation and counted among the flock of those you have chosen. Through Christ our Lord. Amen.” #oxfordoratory Courtesy I recall how, as a child at school, we were sometimes made to learn poetry. At the time I hated it, and baulked at the idea of having to commit to memory The Ancient Mariner, and in fact, never did. But one poem which did touch me, was Belloc’s Courtesy. It began: Of Courtesy, it is much lessThan Courage of Heart or Holiness,Yet in my Walks it seems to meThat the Grace of God is in Courtesy. The poet goes on to tell us how, on one occasion he visited the ‘monks’ at Storrington in Sussex, and that ‘They took me straight into their Hall’where he ‘saw Three Pictures on a wall, And Courtesy was in them all.’ He then tells us the subject of those paintings: The first the Annunciation;The second the Visitation;The third the Consolation,Of God that was Our Lady’s Son. The first was of St. Gabriel;On Wings a-flame from Heaven he fell;And as he went upon one kneeHe shone with Heavenly Courtesy. Our Lady out of Nazareth rode —It was Her month of heavy load;Yet was her face both great and kind,For Courtesy was in Her Mind. The third it was our Little Lord,Whom all the Kings in arms adored;He was so small you could not seeHis large intent of Courtesy. Today is the feast of the Visitation, when we celebrate how our Lady made the long and uncomfortable journey to see her older kinswoman, Elizabeth, and her husband Zachariah, who lived in the hill country of Judah. Courtesy seems to me to be a perfect description of what this moment is all about. Imagine the cordial welcome extended to Mary by the older woman, solicitous for her health and how both women were eager to share with one another the joy of the forthcoming births of their sons. Our Lady had herself gone to visit her older cousin, not simply because she wanted to ‘be there’ for her, but because she wanted to share her own good news, which was not hers alone but for her people and, ultimately for the entire world. This is something Elizabeth seemed to know already! Her enthusiastic greeting is wonderful when she cries out ‘Oh that I should be visited by the Mother of my Lord’. Her unborn son joins in the greeting, kicking and letting his mother know that he too is part of this exciting thing that is happening. Elizabeth’s gracious words: ‘Blessed art thou among women and blessed is the fruit of thy womb’ have been enshrined in our prayer to the Virgin, incorporated with the Angelic Salutation: ‘Hail Mary, full of grace!’ And if today’s feast is about courtesy (so much more than stiff politeness or polished manners, but a warm and real concern for the wellbeing and interest of the other) then it is also about joy. Mary’s presence, together with that of her Divine Son, transformed the home of Elizabeth and Zachariah. ‘Elizabeth was filled with the Holy Spirit’ because of it. St Jose Maria puts it thus: ‘Mary brought joy to her cousin’s home, because she “brought” Christ’. Dame Julian liked to call Jesus ‘our courteous Lord,’ no doubt on account of his gracious dealings with us, flowing from his ‘large intent of Courtesy’! Julian writes in her Showings: And this is a supreme friendship of our courteous Lord, that he protects us so tenderly whilst we are in our sins; and furthermore he touches us most secretly, and shows us our sins by the sweet light of mercy and grace. But when we see ourselves so foul, then we believe that God may be angry with us because of our sins. Then we are moved by the Holy Spirit through contrition to prayer, and we desire with all our might an amendment of ourselves to appease God’s anger, until the time that we find rest of soul and ease of conscience. And then we hope that God has forgiven us our sin; and this is true. And then our courteous Lord shows himself to the soul, happily and with the gladdest countenance, welcoming it as a friend, as if it had been in pain and in prison, saying: My dear darling, I am glad that you have come to me in all your woe. I have always been with you, and now you see me loving, and we are made one in bliss. So sins are forgiven by grace and mercy, and our soul is honourably received in joy, as it will be when it comes into heaven, as often as it comes by the operation of grace of the Holy Spirit and the power of Christ’s Passion. Even though we have just changed all the light bulbs, our church looks its best when we don’t need to use them. #oxfordoratory The flag goes up on feast days! #oxfordoratory Blessings with St Philip’s relic after Vespers on Thursday. #oxfordoratory Look down from heaven, Holy Father, from the loftiness of that mountain to the lowliness of this valley; from that harbour of quietness and tranquillity to this calamitous sea. And now that the darkness of this world hinders no more those benignant eyes of thine from looking clearly into all things, look down and visit, O most diligent keeper, this vineyard which thy right hand planted with so much labour, anxiety and peril. To thee then we fly; from thee we seek for aid; to thee we give our whole selves unreservedly. Thee we adopt for our patron and defender; undertake the cause of our salvation, protect thy clients. To thee we appeal as our leader; rule thine army fighting against the assaults of the devil. To thee, kindest of pilots, we give up the rudder of our lives; steer this little ship of thine, and, placed as thou art on high, keep us off all the rocks of evil desires, that with thee for our pilot and our guide, we may safely come to the port of eternal bliss. #oxfordoratory Our celebrations for St Philip began with First Vespers last night and continue with Masses today. Here St Philip’s relic and altar are incensed during Vespers. #oxfordoratory Our new longer term temporary digital organ was hoisted up to the gallery this week in time for First Vespers of Our Holy Father St Philip. Catch up on our YouTube channel if you missed it, and join us for the Solemn Mass at 6pm this Friday. #oxfordoratory Vessel of the Holy Spirit This year the novena in preparation for the feast of Our Holy Father St Philip corresponds almost exactly with the novena of novenas, the first novena, the nine days of prayer between the Ascension and Pentecost when we wait once more with the Apostles for the outpouring of the Holy Spirit. And this is most appropriate, as St Philip was, more than anything else, a man of the Spirit — as we say in his litany, he is the “vessel of the Holy Spirit”. The story of his life, which we read during the novena, really begins with his singular experience of the Spirit on the Vigil of Pentecost 1544 in the catacomb of St Sebastian when the Spirit descended like a ball of fire, entering his mouth and then into his heart. After that experience — Philip’s own Pentecost — his life would never be the same again. Behind his holiness of life, and his preaching, and his miracles, and his founding of our Institute, there is something fundamental we can learn from St Philip — conveniently for this time in the Church’s liturgical calendar, something about the Holy Spirit. St Philip’s life shows us what happens when we do not simply possess the Holy Spirit, but allow ourselves to be possessed by him. We all have the Holy Spirit dwelling in us — all of us who have been baptised and confirmed. He is in us as much as he was in St Philip Neri, as much as he was in the Apostles at Pentecost. But we are very good at closing ourselves to the Spirit — we, through our sins and our selfishness, our attachments and our self-will, block him out; we ignore his prompting and are deaf to his call, we partition him away into a little forgotten compartment in our souls. Or we make of him a very petty little thing indeed, our God — a God who always agrees with what we say, who wants us to do precisely what we want to do, and makes me right and everyone else wrong. But this is not the Holy Spirit whom we worship; this is not the awesome power of Almighty God, who hovered over the waters at creation, who raised Jesus from the dead, who came down on the Apostles and Our Lady in the Upper Room, who wonderfully penetrated the heart of St Philip, and who gives power and authority to the Church. The Holy Spirit blows where he wills; his ways are not always our ways; and if we give ourselves to him and allow him to possess us completely — as St Philip did — then we will find that he will do more for us than we ask or even imagine; do more through us and with us. St Philip wanted to be a layman — the Spirit called him to the priesthood; he wanted to be a martyr, the Spirit made him a martyr not of blood, but of charity; Philip wanted to preach the Gospel to the pagans in the Indies, the Holy Spirit had other plans. He wanted to be a humble, hidden servant of the Church, living at San Girolamo and doing what good he could, but the Spirit gave him the gift of preaching and teaching and working miracles and reading men’s hearts and winning souls, and brought him to the Vallicella, and through him founded the Congregation of the Oratory, spread, as it now is, across the world. St Philip loved to be unknown — the Holy Spirit moulded and fashioned and transformed him into one of the most popular saints, the Apostle of Rome. St Philip wanted to reform the hearts and souls of those men and women he met — the Spirit used him to reform the very heart of the Church herself. It is not enough to have the Holy Spirit — it is not enough even to think we are serving God or doing great things in his honour. We must allow ourselves to be led by the Spirit, formed and transformed by the Spirit. We must be prepared to give up our own desires and plans — even the good ones — and be taken over by the Holy Spirit to be used for his own purposes. If we do this, if we open up to the Spirit who dwells in our hearts, then the life of St Philip Neri, and indeed of all the saints, shows us that the Spirit will do great things for, in, and through us. St Philip, Vessel of the Holy Spirit and Sweetest of Fathers. Pray for us. A Prayer of Our Holy Father St Philip: My Lord Jesus, I want to love you but you cannot trust me. If you do not help me, I will never do any good. I do not know you; I look for you but I do not find you. Come to me, O Lord. If I knew you, I would also know myself. If I have never loved you before, I want to love you truly now. I want to do your will alone; putting no trust in myself, I hope in you, O Lord. The Paschal Candle has been lit for Masses since the Easter Vigil to represent Christ’s presence among us, as with his disciples for forty days. After the Gospel of the Solemn Mass yesterday, it is extinguished to symbolise his ascension into heaven. #oxfordoratory Look up to heaven On Monday this week some of the Fathers and Brothers spent the day erecting and moving scaffolding — with all due health and safety measures observed — in order to change and improve the lighting on the sanctuary of our church. It is already a great boon for us to see more clearly the beautiful decoration which adorns that focal point of the whole building, as well as being a great help for the priest reading from the missal. Tomorrow as we celebrate the feast of the Ascension, our eyes are directed upwards once more as Our Risen Lord ascends to heaven and for a moment like the Apostles we gaze upwards as he is taken from our sight. How very much more clearly do we see when we make that effort to lift up our eyes towards the Lord. We not only contemplate his glory but from that vantage point are able to see all things in their fullness, seeing them from the perspective of heaven. Prayer, the lifting of the mind and heart to God, enables us to understand and to see clearly, all those other things of life in which we are otherwise immersed. That daily shifting our focus from the things of this world to the vantage point of heaven is that great encouragement for our daily striving for God, so that where Christ goes we too may follow him. It is often an effort for us to pull ourselves out of the daily round in order to lift up our heads and our hearts to the things of heaven, but if we are to have the desire for heaven to drive us on, it is essential that we always keep our goal before us. Not only in prayer, but all those things which form the way we see things must have heaven as their vantage point and aim. Monsignor Knox wrote: In all ages, in all countries, the world acts as a solvent to Catholic piety; breathes an air in which Catholic piety languishes. Man’s intellect always wants to approach things from the human side, from the side from which they can be known by man; shirks and burkes discussion of things from the side of reality, from the side which relates them to God. Man’s art and literary genius is constantly concerned with man at his most human level, his passions, his craven fears, the rebellion of his will against the order in which he lives. All that breathes a poison, for which writers who care about the truth as it is in God have to provide an antidote; they must fight, they must react, but still more they must see things, they must record things, from that higher standpoint which is God’s. You must, sometimes, give the lungs of your soul an airing on these heights, even if the atmosphere of it is more rare, is breathed with more effort, than the other. Or else, the miasma of the modern world will get you down, will weaken your resistance; you will be a prey to the germs of infidelity, to the infection of bad example. And you will forget your Friend. As the Church invites us then on the Ascension to gaze up towards heaven, to contemplate Our Lord, Risen and Ascended, it is perhaps a good time for us to think on the place we give to the things of heaven in our life each day — not only in terms of the time we give to heaven, but also in terms of what we strive for, on what really all our efforts are in fact based. And from our gazing upon heaven, we can then go on like the Apostles, down from that mountain and into the world — to teach the Good News and make disciples of all nations, keeping ever in mind that he whom we meet on those heights of prayer is with us always, even to the end of time. Q: How many Oratorians does it take to change a lightbulb?A: “Change”? Actually the real answer turns out to be three, plus one very helpful volunteer and a lot of scaffolding. The sanctuary lighting is all working at full brightness again! #oxfordoratory Monday 15 May 2023 Congratulations to David who was baptised on Saturday! #oxfordoratory Saturday 13 May 2023 One of this month’s relics on display is St Philip’s own copy of Jacapone da Todi’s “Laude”, which St Philip has written his name in. The story of how it was rediscovered in our church — only days before the first Fathers of the Oratory arrived here from Birmingham — is extraordinary. You might find it hard to believe, unless you heard it straight from the priest who found them, who is now the Bishop of Leeds. Read his account from his St Philip’s day sermon on our website: www.oxfordoratory.org.uk/blog/post/1335-solemnity-of-our-holy-father-saint-philip The novena to St Philip begins on Tuesday after the evening Mass. You will be able to place your own prayer intentions for the novena in St Philip’s chapel over the weekend. There will be Solemn Choral Vespers on Thursday 25 May at 6:30pm. We will welcome Fr Robert Ombres OP from Blackfriars to preach at the Solemn Mass for the feast day itself on Friday 26 May at 6pm. #oxfordoratory Friday 12 May 2023 May Music Sunday 7 MaySolemn Mass 11:005th Sunday of EasterMissa Simile est regnum caelorum LoboJubilate Deo universa terra PalestrinaCaro mea vere est cibus Guerrero Sunday 14 MaySolemn Mass 11:006th Sunday of EasterMissa Regina caeli PalestrinaBenedicite gentes PalestrinaPortio mea White Thursday 18 MaySolemn Mass 18:00The Ascension of the LordMissa Ascendens Christus VictoriaAscendit Deus PhilipsO Rex gloriae Marenzio Sunday 21 MaySolemn Mass 11:007th Sunday of EasterMissa Jam Christus astra ascenderat PalestrinaViri Galilaei PalestrinaEgo sum panis vivus Palestrina Thursday 25 MaySolemn Vespers 18:30Our Holy Father St Philip NeriDeus in adjutorium PadillaMagnificat primi toni VictoriaPangamus Nerio SewellO salutaris hostia LalouxTantum ergo WidorAlleluia GabrieliRespice de caelo Sewell Friday 26 MaySolemn Mass 18:00Our Holy Father St Philip NeriMissa Papae Marcelli PalestrinaIn spiritu humilitatis CroceJubilate Deo a8 G Gabrieli Sunday 28 MaySolemn Mass 11:00PentecostMissa De la Batalla GuerreroConfirma hoc deus LassusLoquebantur variis linguis Palestrina The Oratory 25 Woodstock Road © 2023 Oxford Oratory. All rights reserved. The Oxford Oratory of St Philip Neri is a Registered Charity number 1018455	https://www.oxfordoratory.org.uk/saints/blog/post/9881-praying-with-words/blog/post/9898-9898/giving.php
Global Natural Language Processing NLP in Healthcare Market Research Report 2023(Status and Outlook) Global Natural Language Processing NLP in Healthcare Market Research Report 2023(Status and Outlook) Global Natural Language Processing NLP in Healthcare Market Research Report 2023(Status and - Market research report and industry analysis - 34156519 Global Natural Language Processing NLP in Healthcare Market Research Report 2023(Status and Outlook) Best Price Guarantee Price from $2,800 Length 110 Pages Publisher Bosson Research Published Date May, 2023 SKU BOSS17934493 Table of Contents Description Global Natural Language Processing NLP in Healthcare Market Research Report 2023(Status and Outlook) Report Overview Natural language processing (NLP) is a branch of artificial intelligence that deals with the interaction between computers and humans using the natural language (spoken or written data), not in the artificial languages such as Java and C++. The Global Natural Language Processing NLP in Healthcare Market Size was estimated at USD 851.80 million in 2022 and is projected to reach USD 3070.00 million by 2029, exhibiting a CAGR of 20.10% during the forecast period. Bosson Research’s latest report provides a deep insight into the global Natural Language Processing NLP in Healthcare market covering all its essential aspects. This ranges from a macro overview of the market to micro details of the market size, competitive landscape, development trend, niche market, key market drivers and challenges, SWOT analysis, Porter’s five forces analysis, value chain analysis, etc. The analysis helps the reader to shape the competition within the industries and strategies for the competitive environment to enhance the potential profit. Furthermore, it provides a simple framework for evaluating and accessing the position of the business organization. The report structure also focuses on the competitive landscape of the Global Natural Language Processing NLP in Healthcare Market, this report introduces in detail the market share, market performance, product situation, operation situation, etc. of the main players, which helps the readers in the industry to identify the main competitors and deeply understand the competition pattern of the market. In a word, this report is a must-read for industry players, investors, researchers, consultants, business strategists, and all those who have any kind of stake or are planning to foray into the Natural Language Processing NLP in Healthcare market in any manner. Global Natural Language Processing NLP in Healthcare Market: Market Segmentation Analysis The research report includes specific segments by region (country), manufacturers, Type, and Application. Market segmentation creates subsets of a market based on product type, end-user or application, Geographic, and other factors. By understanding the market segments, the decision-maker can leverage this targeting in the product, sales, and marketing strategies. Market segments can power your product development cycles by informing how you create product offerings for different segments. Key Company 3M Nuance Communications SAS IBM Microsoft Corporation Averbis Health Fidelity Dolbey Systems Market Segmentation (by Type) Machine Translation Information Extraction Automatic Summarization Text and Voice Processing Other Natural Language Processing (NLP) Market Segmentation (by Application) Blood Cancer Breast Cancer Gastrointestinal Cancer Prostate Cancer Respiratory or Lung Cancer Others Geographic Segmentation Europe (Germany, UK, France, Russia, Italy, Rest of Europe) Asia-Pacific (China, Japan, South Korea, India, Southeast Asia, Rest of Asia-Pacific) South America (Brazil, Argentina, Columbia, Rest of South America) The Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, South Africa, Rest of MEA) Key Benefits of This Market Research: Industry drivers, restraints, and opportunities covered in the study Neutral perspective on the market performance Recent industry trends and developments Competitive landscape & strategies of key players Potential & niche segments and regions exhibiting promising growth covered Historical, current, and projected market size, in terms of value In-depth analysis of the Natural Language Processing NLP in Healthcare Market Overview of the regional outlook of the Natural Language Processing NLP in Healthcare Market: Key Reasons to Buy this Report: Access to date statistics compiled by our researchers. These provide you with historical and forecast data, which is analyzed to tell you why your market is set to change This enables you to anticipate market changes to remain ahead of your competitors You will be able to copy data from the Excel spreadsheet straight into your marketing plans, business presentations, or other strategic documents The concise analysis, clear graph, and table format will enable you to pinpoint the information you require quickly Provision of market value (USD Billion) data for each segment and sub-segment Indicates the region and segment that is expected to witness the fastest growth as well as to dominate the market Analysis by geography highlighting the consumption of the product/service in the region as well as indicating the factors that are affecting the market within each region Competitive landscape which incorporates the market ranking of the major players, along with new service/product launches, partnerships, business expansions, and acquisitions in the past five years of companies profiled The current as well as the future market outlook of the industry concerning recent developments which involve growth opportunities and drivers as well as challenges and restraints of both emerging as well as developed regions Includes in-depth analysis of the market from various perspectives through Porter’s five forces analysis Provides insight into the market through Value Chain Market dynamics scenario, along with growth opportunities of the market in the years to come 6-month post-sales analyst support Chapter Outline Chapter 1 mainly introduces the statistical scope of the report, market division standards, and market research methods. Chapter 2 is an executive summary of different market segments (by region, product type, application, etc), including the market size of each market segment, future development potential, and so on. It offers a high-level view of the current state of the Natural Language Processing NLP in Healthcare Market and its likely evolution in the short to mid-term, and long term. Chapter 3 makes a detailed analysis of the market's competitive landscape of the market and provides the market share, capacity, output, price, latest development plan, merger, and acquisition information of the main manufacturers in the market. Chapter 4 is the analysis of the whole market industrial chain, including the upstream and downstream of the industry, as well as Porter's five forces analysis. Chapter 5 introduces the latest developments of the market, the driving factors and restrictive factors of the market, the challenges and risks faced by manufacturers in the industry, and the analysis of relevant policies in the industry. Chapter 6 provides the analysis of various market segments according to product types, covering the market size and development potential of each market segment, to help readers find the blue ocean market in different market segments. Chapter 7 provides the analysis of various market segments according to application, covering the market size and development potential of each market segment, to help readers find the blue ocean market in different downstream markets. Chapter 8 provides a quantitative analysis of the market size and development potential of each region and its main countries and introduces the market development, future development prospects, market space, and capacity of each country in the world. Chapter 9 introduces the basic situation of the main companies in the market in detail, including product sales revenue, sales volume, price, gross profit margin, market share, product introduction, recent development, etc. Chapter 10 provides a quantitative analysis of the market size and development potential of each region in the next five years. Chapter 11 provides a quantitative analysis of the market size and development potential of each market segment (product type and application) in the next five years. Chapter 12 is the main points and conclusions of the report. 1 Research Methodology and Statistical Scope 1.1 Market Definition and Statistical Scope of Natural Language Processing NLP in Healthcare 1.2 Key Market Segments 1.2.1 Natural Language Processing NLP in Healthcare Segment by Type 1.2.2 Natural Language Processing NLP in Healthcare Segment by Application 1.3 Methodology & Sources of Information 1.3.1 Research Methodology 1.3.2 Research Process 1.3.3 Market Breakdown and Data Triangulation 1.3.4 Base Year 1.3.5 Report Assumptions & Caveats 2 Natural Language Processing NLP in Healthcare Market Overview 2.1 Global Natural Language Processing NLP in Healthcare Market Size (M USD) Estimates and Forecasts (2018-2029) 2.2 Market Segment Executive Summary 2.3 Global Market Size by Region 3 Natural Language Processing NLP in Healthcare Market Competitive Landscape 3.1 Global Natural Language Processing NLP in Healthcare Revenue Market Share by Manufacturers (2018-2023) 3.2 Natural Language Processing NLP in Healthcare Market Share by Company Type (Tier 1, Tier 2, and Tier 3) 3.3 Manufacturers Natural Language Processing NLP in Healthcare Sales Sites, Area Served, Service Type 3.4 Natural Language Processing NLP in Healthcare Market Competitive Situation and Trends 3.4.1 Natural Language Processing NLP in Healthcare Market Concentration Rate 3.4.2 Global 5 and 10 Largest Natural Language Processing NLP in Healthcare Players Market Share by Revenue 3.4.3 Mergers & Acquisitions, Expansion 4 Natural Language Processing NLP in Healthcare Value Chain Analysis 4.1 Natural Language Processing NLP in Healthcare Value Chain Analysis 4.2 Midstream Market Analysis 4.3 Downstream Customer Analysis 5 The Development and Dynamics of Natural Language Processing NLP in Healthcare Market 5.1 Key Development Trends 5.2 Driving Factors 5.3 Market Challenges 5.4 Market Restraints 5.5 Industry News 5.5.1 Mergers & Acquisitions 5.5.2 Expansions 5.5.3 Collaboration/Supply Contracts 5.6 Industry Policies 6 Natural Language Processing NLP in Healthcare Market Segmentation by Type 6.1 Evaluation Matrix of Segment Market Development Potential (Type) 6.2 Global Natural Language Processing NLP in Healthcare Market Size Market Share by Type (2018-2023) 6.3 Global Natural Language Processing NLP in Healthcare Sales Growth Rate by Type (2019-2023) 7 Natural Language Processing NLP in Healthcare Market Segmentation by Application 7.1 Evaluation Matrix of Segment Market Development Potential (Application) 7.2 Global Natural Language Processing NLP in Healthcare Market Size (M USD) by Application (2018-2023) 7.3 Global Natural Language Processing NLP in Healthcare Sales Growth Rate by Application (2019-2023) 8 Natural Language Processing NLP in Healthcare Market Segmentation by Region 8.1 Global Natural Language Processing NLP in Healthcare Market Size by Region 8.1.1 Global Natural Language Processing NLP in Healthcare Market Size by Region 8.1.2 Global Natural Language Processing NLP in Healthcare Market Share by Region 8.2 North America 8.2.1 North America Natural Language Processing NLP in Healthcare Market Size by Country 8.2.2 U.S. 8.2.3 Canada 8.2.4 Mexico 8.3 Europe 8.3.1 Europe Natural Language Processing NLP in Healthcare Market Size by Country 8.3.2 Germany 8.3.3 France 8.3.4 U.K. 8.3.5 Italy 8.3.6 Russia 8.4 Asia Pacific 8.4.1 Asia Pacific Natural Language Processing NLP in Healthcare Market Size by Region 8.4.2 China 8.4.3 Japan 8.4.4 South Korea 8.4.5 India 8.4.6 Southeast Asia 8.5 South America 8.5.1 South America Natural Language Processing NLP in Healthcare Market Size by Country 8.5.2 Brazil 8.5.3 Argentina 8.5.4 Columbia 8.6 Middle East and Africa 8.6.1 Middle East and Africa Natural Language Processing NLP in Healthcare Market Size by Region 8.6.2 Saudi Arabia 8.6.3 UAE 8.6.4 Egypt 8.6.5 Nigeria 8.6.6 South Africa 9 Key Companies Profile 9.1 3M 9.1.1 3M Natural Language Processing NLP in Healthcare Basic Information 9.1.2 3M Natural Language Processing NLP in Healthcare Product Overview 9.1.3 3M Natural Language Processing NLP in Healthcare Product Market Performance 9.1.4 3M Business Overview 9.1.5 3M Natural Language Processing NLP in Healthcare SWOT Analysis 9.1.6 3M Recent Developments 9.2 Linguamatics 9.2.1 Linguamatics Natural Language Processing NLP in Healthcare Basic Information 9.2.2 Linguamatics Natural Language Processing NLP in Healthcare Product Overview 9.2.3 Linguamatics Natural Language Processing NLP in Healthcare Product Market Performance 9.2.4 Linguamatics Business Overview 9.2.5 Linguamatics Natural Language Processing NLP in Healthcare SWOT Analysis 9.2.6 Linguamatics Recent Developments 9.3 Amazon AWS 9.3.1 Amazon AWS Natural Language Processing NLP in Healthcare Basic Information 9.3.2 Amazon AWS Natural Language Processing NLP in Healthcare Product Overview 9.3.3 Amazon AWS Natural Language Processing NLP in Healthcare Product Market Performance 9.3.4 Amazon AWS Business Overview 9.3.5 Amazon AWS Natural Language Processing NLP in Healthcare SWOT Analysis 9.3.6 Amazon AWS Recent Developments 9.4 Nuance Communications 9.4.1 Nuance Communications Natural Language Processing NLP in Healthcare Basic Information 9.4.2 Nuance Communications Natural Language Processing NLP in Healthcare Product Overview 9.4.3 Nuance Communications Natural Language Processing NLP in Healthcare Product Market Performance 9.4.4 Nuance Communications Business Overview 9.4.5 Nuance Communications Recent Developments 9.5 SAS 9.5.1 SAS Natural Language Processing NLP in Healthcare Basic Information 9.5.2 SAS Natural Language Processing NLP in Healthcare Product Overview 9.5.3 SAS Natural Language Processing NLP in Healthcare Product Market Performance 9.5.4 SAS Business Overview 9.5.5 SAS Recent Developments 9.6 IBM 9.6.1 IBM Natural Language Processing NLP in Healthcare Basic Information 9.6.2 IBM Natural Language Processing NLP in Healthcare Product Overview 9.6.3 IBM Natural Language Processing NLP in Healthcare Product Market Performance 9.6.4 IBM Business Overview 9.6.5 IBM Recent Developments 9.7 Microsoft Corporation 9.7.1 Microsoft Corporation Natural Language Processing NLP in Healthcare Basic Information 9.7.2 Microsoft Corporation Natural Language Processing NLP in Healthcare Product Overview 9.7.3 Microsoft Corporation Natural Language Processing NLP in Healthcare Product Market Performance 9.7.4 Microsoft Corporation Business Overview 9.7.5 Microsoft Corporation Recent Developments 9.8 Averbis 9.8.1 Averbis Natural Language Processing NLP in Healthcare Basic Information 9.8.2 Averbis Natural Language Processing NLP in Healthcare Product Overview 9.8.3 Averbis Natural Language Processing NLP in Healthcare Product Market Performance 9.8.4 Averbis Business Overview 9.8.5 Averbis Recent Developments 9.9 Health Fidelity 9.9.1 Health Fidelity Natural Language Processing NLP in Healthcare Basic Information 9.9.2 Health Fidelity Natural Language Processing NLP in Healthcare Product Overview 9.9.3 Health Fidelity Natural Language Processing NLP in Healthcare Product Market Performance 9.9.4 Health Fidelity Business Overview 9.9.5 Health Fidelity Recent Developments 9.10 Dolbey Systems 9.10.1 Dolbey Systems Natural Language Processing NLP in Healthcare Basic Information 9.10.2 Dolbey Systems Natural Language Processing NLP in Healthcare Product Overview 9.10.3 Dolbey Systems Natural Language Processing NLP in Healthcare Product Market Performance 9.10.4 Dolbey Systems Business Overview 9.10.5 Dolbey Systems Recent Developments 10 Natural Language Processing NLP in Healthcare Regional Market Forecast 10.1 Global Natural Language Processing NLP in Healthcare Market Size Forecast 10.2 Global Natural Language Processing NLP in Healthcare Market Forecast by Region 10.2.1 North America Market Size Forecast by Country 10.2.2 Europe Natural Language Processing NLP in Healthcare Market Size Forecast by Country 10.2.3 Asia Pacific Natural Language Processing NLP in Healthcare Market Size Forecast by Region 10.2.4 South America Natural Language Processing NLP in Healthcare Market Size Forecast by Country 10.2.5 Middle East and Africa Forecasted Consumption of Natural Language Processing NLP in Healthcare by Country 11 Forecast Market by Type and by Application (2024-2029) 11.1 Global Natural Language Processing NLP in Healthcare Market Forecast by Type (2024-2029) 11.2 Global Natural Language Processing NLP in Healthcare Market Forecast by Application (2024-2029) 12 Conclusion and Key Findings Global Natural Language Processing (NLP) in Healthcare Market Growth (Status and Outlook) 2023-2029 January, 2023 \| Published by: LP Information, Inc. \| USD $3,660 Natural Language Processing (NLP) March, 2023 \| Published by: Global Industry Analysts \| USD $5,450 The 2023-2028 World Outlook for Healthcare NLP (Natural Language Processing) May, 2022 \| Published by: Icon Group International, Inc. \| USD $995 Natural Language Processing (NLP) in Healthcare and Life Sciences April, 2023 \| Published by: Global Industry Analysts \| USD $5,600 Natural Language Processing NLP Market June, 2022 \| Published by: Future Market Insights \| USD $5,000 Global Natural language processing (NLP) Market Growth (Status and Outlook) 2023-2029 March, 2023 \| Published by: LP Information, Inc. \| USD $3,660 34156520 PDF E-mail From Publisher: $2,800 Global Site License Fulfilled by Publisher: $5,600	https://www.marketresearch.com/Bosson-Research-v4252/Global-Natural-Language-Processing-NLP-34156519/
Where's the sugar? \| Bakingbusiness.com \| May 20, 2016 20:16 \| Baking Business Complete resource for grain-based food industry news, commodity markets, ingredients, processing trends and innovations, publications and more. Where's the sugar? Users want more cane sugar, but total supplies seen as adequate. The U.S. Department of Agriculture, which administers the U.S. sugar program, is facing a conundrum. Sugar users citing increased cane sugar demand and some refiners citing tight raw cane supplies are seeking increased raw cane imports while beet sugar supplies are readily available. An increase in raw cane imports would displace domestic beet sugar, potentially creating an overall sugar oversupply and pushing beet processors into forfeitures. On May 5 a group of 45 U.S. Senators and Representatives in a letter requested U.S. Secretary of Agriculture Tom Vilsack “immediately” use his authority to make adequate sugar supplies available at reasonable prices. The U.S.D.A. has long said it will act “cautiously” when deciding to increase the tariff-rate quota (T.R.Q.), which may be done from April 1 to the end of the marketing year Sept. 31 without invoking an “emergency” clause. At the same time, the U.S.D.A. is mandated by Congress to run the sugar program at no cost to U.S. taxpayers, which it usually does, although opponents contend U.S. consumers (industrial and retail) pay higher prices for sugar due to restrictions that limit lower cost imports. But it’s not that simple. First, not all cane refiners are short of raw sugar. It is mainly those who depend heavily on imported raws that are experiencing a shortage. Those with domestic sources of raw sugar are not seeking additional raw cane imports and at least one of those has confirmed it has refined cane sugar available. Some in the trade contend the refiners seeking additional raw cane imports are in an oversold position and can’t deliver on contracted refined cane sugar sales, although it should be noted those refiners must run well below capacity without adequate imports. Supporting the side for increased raw cane imports is apparent increasing demand for non-bioengineered cane sugar over beet sugar, which is almost entirely from bioengineered seed. The U.S.D.A. a month or so back noted increased demand for cane and sagging demand for beet sugar, but also noted the rate of increase for cane was slowing and the rate of decrease for beet also was slowing. The Hershey Co. was the first and most visible major sugar user to announce it would use only non-bioengineered sugar. A few others have followed and there’s been lots of talk, in part prompted by the labeling law in Vermont that goes into effect July 1 requiring foods containing bioengineered ingredients be labeled as such. One major beet sugar producer conducted its own study on the level of switching from bioengineered beet sugar to non-bioengineered cane sugar and determined the overall shift at the current time was not as great as indicated by the amount of press coverage concerning the debate. Many in the trade expect beet sugar to regain some demand as the price spread between beet and cane sugar widens. Historically cane sugar has traded about 1c to 2c a lb above beet sugar; that premium has widened to as much as 4c a lb in some markets. Another twist was added May 10 when the U.S.D.A. projected 2016-17 (beginning Oct. 1) U.S. beet sugar production to increase 0.5% to a record 5.09 million tons and cane sugar output to decrease 6.4% from this year. Despite the decrease in domestic cane sugar, higher imports next year, mainly from Mexico, will result in the total supply of cane sugar nearly the same as this year. The increase in beet sugar may indicate producers aren’t overly concerned about losing more demand to cane sugar. On May 5, the U.S.D.A. also announced the 2016-17 raw sugar T.R.Q. at the minimum required under World Trade Organization agreements and unchanged from 2015-16, and a 22,046-ton increase in the 2015-16 specialty sugar T.R.Q. (not raws), which disappointed sugar users and select refiners who had been seeking as much as a 500,000-ton increase in the imported raw T.R.Q. So the U.S.D.A. in its administration of the U.S. sugar program has its work cut out in determining if it can increase raw cane imports without throwing the total market into oversupply. It would appear, at least at this point, that total sugar supplies are adequate under the current rules in which the U.S.D.A. must work, which would preclude an increase in the raw sugar T.R.Q. Ingredient Markets Sugar	https://www.bakingbusiness.com/articles/30280-where-s-the-sugar
Railway Catering — Past and Present Methods \| NZETC Railway Catering — Past and Present Methods Railway Catering Past and Present Methods Until the latter part of 1917 Railway Catering in New Zealand was conducted in dining cars on the principal long distance express trains and in privately leased refreshment rooms for the less important trains. The dining car system, quite apart from the financial loss at which it was operated, was not at all satisfactory from the viewpoint of either the Department or the majority of the travelling public. The seating accommodation of each car was limited to 24, and as the cooking and galley facilities were also limited, one can readily appreciate the difficulties attendant upon serving several sittings and at the same time maintaining a desirable standard for the meals served. In many instances it was found impossible to cater for all who desired to partake of meals. Most of the dishes required during the journey were cooked off either in land kitchens or in the dining cars some considerable time prior to the commencement of the meals, which naturally affected the standard. The difficulties connected with the working of the cars were considerable and resulted in passengers suffering much discomfort and inconvenience in travelling backwards and forwards through long trains to reach the dining car and this discomfort was very severe in cold, wet weather. One way to have overcome some of the inconvenience would have been the provision of a second dining car, but as this would, in most cases, have meant the loss of a passenger car on account of haulage limitation, such a step was considered inadvisable. In any case, such relief would have been only temporary, as the traffic throughout has increased. The alternative method of effecting the desired and permanent improvement was found in an entire change in the Department's policy, and the decisions reached in this respect included:- 1.The immediate discontinuance of the dining car service. 2.The establishment of a railway refreshment branch. 3.The taking over and immediate control by Department of eight privately leased refreshment rooms. 4.The ultimate control of the refreshment room service over the whole system, as and when opportunities and circumstances warranted. The far reaching effects of such a drastic change in policy will be appreciated, and not the least of these included arranging for all meals and refreshments to be provided in refreshment rooms at which adequate train stops had to be provided. The internal fittings of the dining cars were dismantled and the cars refitted as passenger cars for the exclusive use of ladies and children on express trains. With the inauguration of the new policy it became possible to immediately set and maintain a much higher standard as regards both quality and service and at the same time provide greater variety of items than was possible when the dining cars were running. Owing to the widely divergent views held by different members of the community as to what constitutes a satisfactory standard, or sufficiency, of the meal in order that the inner-man may be satisfied, it would, of course, be impossible to please everyone no matter what system was in vogue. The almost entire absence of complaints, however, leads us to conclude that the Department's efforts are appreciated and meet with the approval of the great majority of its customers. One of the first serious obstacles to be overcome by the newly constituted refreshment branch under the control of Mr. Irwin Faris , was connected with the general construction of the refreshment room buildings and it was found that in some cases entirely new buildings were required whilst in others alterations and additions to existing buildings sufficed. As already indicated, the dining cars were unprofitable, and in this respect the refreshment room system is in marked contrast. The Branch is run on purely commercial lines and is required to meet all legitimate trading charges accruing in the conduct of the business and some of these include rent of refreshment rooms and offices, interest on capital invested in stocks, depreciation, and railway freights. The policy of ultimately assuming control of all the refreshment rooms is being gradually given effect to and twenty-five refreshment rooms, also the catering on the Lake Wakatipu steamers, are now under Departmental control. The revenue of the Branch has increased from £49,702 for the year ended 31st March, 1919 (the first complete year) to £133,339 for the year just ended. The staff employed at 31st March, 1918, numbered 117, whilst at 31st March, 1926, the number stood at 363. The latest refreshment rooms erected by the Department are commodious structures, well furnished and fitted, thus enabling customers to obtain their requirements with promptitude and a minimum of inconvenience. The provision of modern, and up-to-date equipment for the assistants with adequate facilities for the preparation and preliminary work, enables the organisation of the rooms to be run without hitch. It is proposed, in a future article, to give a descriptive account of the internal organisation, fittings and equipment of both dining and counter rooms. All items sold at the Department's refreshment rooms are of standard size and as far as is practicable, of standard quality. Bakeries have been erected at Frankton Junction, Marton and Oamaru rooms, where qualified tradesmen are in charge. The bakeries turn out the bread and small goods required by the refreshment rooms situated within convenient distances; such supplies being conveyed in suitable hampers fitted with trays. N.Z. Railways Refreshment Room, Marton Waitresses-in-charge of refreshment rooms not served from the bakeries are required, before being placed in charge of a room, to become thoroughly qualified in the making and baking of pies, small goods, etc., to the standard set. Responsible officers of the Department who have travelled in other countries are unanimous in stating that the refreshment room standard and service on our system-having due regard for the tariff charges, cost of commodities, wages, etc.-is equal to anything met with during their travels, and the latest officer to affirm this is Mr. J. Pickard, who has recently returned from abroad. Cadet, in Manager's office, took off message “No 111 delayed one hour. Injector on engine refused to work.” Cadet suggested to operator that he supply injectors' names as it would probably be a case for suspension. * * * The noblest contribution which any man can make for the benefit of posterity is that of a good character.-Winthrop. * * * You cannot dream yourself into a character, you must hammer and forge yourself one.- Froude.	https://nzetc.victoria.ac.nz/tm/scholarly/tei-Gov01_03Rail-t1-body-d15.html
Advertorial: Innovators of Hyaluronic Acid - Aesthetics Advertorial: Innovators of Hyaluronic Acid 14 Jun 2023 Share this article Discover the history of IBSA with HA Derma From its humble beginnings, IBSA is a large multinational pharmaceutical company present in more than 90 countries across five continents, with 17 subsidiaries. It has more than 2,000 collaborators, 90 patent families and products covering 10 therapeutic areas including cosmetic dermatology with Profhilo®, Aliaxin® and Viscoderm®. Founded in 1945, IBSA started life as a small pharmaceutical company in Lugano, Switzerland. By 1985, it was acquired by the current CEO and Group President Arturo Licenziati. His vision saw IBSA’s revival and growth into the 1990s, thanks to the production of a treatment for osteoarthritis which saw turnover increase and paved the way for a new manufacturing plant. Becoming a world leader With a desire to expand into other markets, Arturo entered a partnership with Laboratoires Genevrier in 1988, now the French subsidiary, IBSA Pharma SAS, followed by the establishment of IBSA Farmaceutici Italia SrL in 1992, which includes the dermo-aesthetic division, IBSA Derma. Following a fire at its factory in 1999, IBSA turned the event into a positive with the construction of manufacturing plants and a pivot into a new field of research, making IBSA one of the world’s leading companies in reproductive medicine. In these early years, IBSA proved it is a company that looks to the future and is ready to face any challenge; a company that pushes the limits of its possibilities further and is willing to take paths yet untravelled. From the turn of the millennium, new factories and production plants were established in Switzerland, Italy and China, and specialised expertise was sought with increased hiring of well-trained and motivated collaborators. This led to the development of cutting-edge technologies and innovative pharmaceutical products. Development of HA products Of particular importance was IBSA’s investment and partnership with Altergon Italia in 2002. This ensured that the group could supply high-quality transdermal patches and injectable hyaluronic acid (HA) products based on the Hydrolift® action concept. The development and 2016 launch of Profhilo® – a bio-remodelling solution of co-operative hybrid complexes of ultra-pure, thermally stabilised HA developed using IBSA’s patented hybrid NAHYCO® technology. Sustainability is key IBSA’s journey into the future is one that harnesses sustainability integrated into all aspects of its social, environmental and economic strategies, and began in 2015 with the publication of its first Sustainability Report. By 2021, IBSA expounded the company’s desire to be present not only in the therapeutic field, but also as a promoter of wellbeing and innovation, leading to the IBSA – Close to you concept, a commitment to creating value for future generations. The first project was centralising IBSA activities at COSMOS, a new site located in CorPharma, Lugano, its new industrial district whose heart is social sustainability and community. With a total area equal to three football fields, COSMOS is IBSA’s largest production plant, designed to be a beautiful, innovative, technological and sustainable building. COSMOS and CorPharma create a space in which to live and work well, meaning IBSA can create conditions that improve productivity, quality of work and atmosphere. For IBSA, sustainability means operating responsibly to generate economic value, assigning the same level of priority to the promotion of social progress and the conservation of environmental resources. IBSA distribution model The individual complexities and challenges of different markets can benefit greatly from the expertise (and strategic partnership) of a local distributor. With a vast product portfolio available for a global market, IBSA operates individual distribution models in each of their territories. These can range from direct distribution across the whole IBSA portfolio across 10 therapeutic areas (ranging from pharma to aesthetics), to independent distributors representing all therapeutic areas, or a combination of a direct IBSA subsidiary combined with independent distributor in one market. The UK is an example of a successful combination model when the offices of IBSA Pharma UK were established in 2022 after acquisition of the local distributor of IBSA’s fertility drugs. The IBSA aesthetic portfolio continues to be successfully retained by independent distributor HA-Derma whose success in the UK & Ireland has had a global impact, and their accomplishment continues to attain recognition, winning numerous IBSA’s distributors awards to date. For more information on IBSA’s portfolio of products, access to full sustainability reports and others, please visit www.ibsagroup.com This advertorial was written and supplied by HA Derma Ltd. From its humble beginnings, IBSA is a large multinational pharmaceutical company present in more than 90 countries across five continents, with 17 subsidiaries. It has more than 2,000 collaborators, 90 patent families and products covering 10 therapeutic areas including cosmetic dermatology with Profhilo®, Aliaxin® and Viscoderm®. Founded in 1945, IBSA started life as a small pharmaceutical company in Lugano, Switzerland. By 1985, it was acquired by the current CEO and Group President Arturo Licenziati. His vision saw IBSA’s revival and growth into the 1990s, thanks to the production of a treatment for osteoarthritis which saw turnover increase and paved the way for a new manufacturing plant. Upgrade to become a Full Member to read all of this article. Subscribe now	https://aestheticsjournal.com/feature/advertorial-innovators-of-hyaluronic-acid
Photo and Mechano-luminescence properties of novel Zn 1-x Eu x Al 2-y Ba y O 4 advance blue phosphor \| Request PDF Request PDF \| Photo and Mechano-luminescence properties of novel Zn 1-x Eu x Al 2-y Ba y O 4 advance blue phosphor \| In this work, we have synthesized novel and efficient material Zn 0.9655 Ba 0.0345 Al 1.9655 O 4 :Eu 0.0345 phosphors by low temperature (500 • C)... \| Find, read and cite all the research you need on ResearchGate Photo and Mechano-luminescence properties of novel Zn 1-x Eu x Al 2-y Ba y O 4 advance blue phosphor November 2020 Physica B Condensed Matter 602(8) DOI: 10.1016/j.physb.2020.412612 Request full-text Abstract In this work, we have synthesized novel and efficient material Zn 0.9655 Ba 0.0345 Al 1.9655 O 4 :Eu 0.0345 phosphors by low temperature (500 • C) combustion route, and for better crystallinity, we have annealed with 850-1350 • C in reducing atmosphere of 15% of H 2 /85% of N 2. The crystal structural, element mapping and distribution, morphology, particle homogeneity, and optical properties were measured by various spectroscopic techniques. Photoluminescence (PL) spectra were monitored by excitation and emission (λ ex. = 492 nm, and λ em. = 354 nm) spectra. The PL spectra of phosphors have recorded in order to assess various samples. The PL intensive emission was found at 492 nm due to the Eu (II) ions. The obtained zinc aluminate phosphors have found higher lumi-nescence efficiency due to the high purity and perfect spinel crystallinity of the material. The maximum quantum efficiency was 93.2%, the FWHM was ~64 nm, and the colour purity was 50% have measured for sample S6. With the PL spectrum, we have found good Mechanoluminescence (ML) optical properties of the S6 sample. Therefore, S6 phosphor would be great and useful material for blue pigment, blue LEDs phosphors as well as may be used in various technology in solid-state lighting. ... • The SEM images in Fig. 4 Table 2 the XRD values are similar to the XRD values in Table 3 of a previously published article [1]. • The reported peak angles, FWHM, Crystalline sizes and the d-spacing are the same as those that have been presented in a previously published article [2] . • In Fig. 8 Fig. 6 in a previously published article [2]. ... ... • The reported peak angles, FWHM, Crystalline sizes and the d-spacing are the same as those that have been presented in a previously published article [2]. • In Fig. 8 Fig. 6 in a previously published article [2] . • In Fig. 9 the Raman spectrum is similar to Fig. 7 of a previously published article [2]. ... ... • In Fig. 8 Fig. 6 in a previously published article [2]. • In Fig. 9 the Raman spectrum is similar to Fig. 7 of a previously published article [2] . • Fig. 10 appears to be highly similar to Fig. 10 in a previously published article [3]. • Fig. 17B and C appear to overlap with Fig. 15A and D from a previously published article [4]. ... Retraction Note to: Investigation of structural, luminescence, and anti-bacterial properties of novel Zn1−xEuxAl2−yO4Sry phosphor Article Full-text available Akshkumar kumar Verma D. P. Bisen Shubham Nema Arun kumar Singh ... The crystal structure, phase purity, crystallite size, and micro-strain of the Zn 1-x Sr y Al 2-y O 4 :Eu x powder phosphor were examined carefully by XRD diffractograms as shown in Fig. 1. All peaks of Zn 1-x Sr 0.05 Al 1.95 O 4 :Eu x (x = 0.01, 0.02, 0.03, 0.04, 0.05, 0.06) are recorded in the XRD pattern. ... ... The crystal structure, phase purity, crystallite size, and micro-strain of the Zn 1-x Sr y Al 2-y O 4 :Eu x powder phosphor were examined carefully by XRD diffractograms as shown in Fig. 1. All peaks of Zn 1-x Sr 0.05 Al 1.95 O 4 :Eu x (x = 0.01, 0.02, 0.03, 0.04, 0.05, 0.06) are recorded in the XRD pattern. The very less amount of doped Europium (II) and Strontium (II) ions does not create a crystal defect in phase composition. ... ... It indicates that irregular morphology, homogenous elemental mixing, and unequal micro-nano crystallites (agglomerated) of synthesized phosphor sample (ZAS-5) have agglomerates with irregular morphology. Also, crystallites are obtained by FESEM images similar to the results obtained from the XRD pattern, showing that [4, 30,39,40]. The average crystallite size or average grain size (D) was calculated using the lineal intercept technique (LIT) according to Eq. (5) [41]. ... Investigation of structural, luminescence, and anti-bacterial properties of novel Zn1−xEuxAl2−yO4Sry phosphor Article Full-text available Jun 2022 J MATER SCI-MATER EL Akshkumar kumar Verma D.P. Bisen Shubham Nema Arun kumar Singh In this research work, we synthesized novel zinc aluminate (Zn1−xEuxAl2−yO4Sry) sky bluish phosphors at 500 °C via the combustion route method, and for better crystallinity, we were annealed in condensing envelope with 85% of N2 and 15% of H2 at 1200 °C. The spinel cubic phase, elemental mapping elements, surface morphology, and vibrational properties were observed by powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier Transform Infrared), and Raman spectroscopic techniques, respectively. The photoluminescence (PL) spectra were monitored by λex. = 490 nm (excitation wavelength) and λem. = 392 (emission wavelength). Due to Eu²⁺ ions at 490 nm, intense photoluminescence spectra have been found. The PL efficiency has been found for synthesized Zn0.95Eu0.05Al1.95O4Sr0.05 (ZAS-5) phosphor at around 93.2%. The high Corelated Color Temperature (~ 25,963 K), low color purity (50%), and low Color Rendering Index (~ 44) value were found for the ZAS-5 sample, that result (temperature) is indicating the prepared phosphor is emitting cold sky bluish light which may be useful for a sleeping light lamp, activation of photosynthesis reaction for crops, and background color for the display screen, because it may be a low dangerous color for human eyes. Antibacterial activity of phosphors indicated that combustion derived synthesized all ZAS samples may behave just like as an eco-material. Observation of up conversion/ down conversion luminescence and structural analysis of La2Zr2O7: Pr3+ nano phosphors Article Sep 2022 MAT SCI SEMICON PROC Amalesh Kumar J. Manam Charge transfer band (CTB) transition significantly improves the photoluminescence properties of phosphor materials due to its higher absorption efficiency than the characteristic excitations of rare-earth ions. La2Zr2O7 (LZO): Pr³⁺ phosphors with varying doping concentrations of Pr³⁺ were synthesized through the hydrothermal method. The formation of the pure pyrochlore phase was confirmed from powder X-ray diffraction (XRD). The structural analysis was performed from Rietveld refinement of the XRD data. Field emission electron microscope (FESM) equipped with EDX spectroscopy was used to study the surface morphology and elemental compositions. X-ray photoelectron spectroscopy (XPS) analysis of the optimized sample provides the bonding state of the compositions. Under 257 nm CTB excitation, the photoluminescence emission spectra (PL) exhibit all the characteristic emission peaks of Pr³⁺ ion. The transitions involved in the excitation and emission process have been shown in the energy level diagram of Pr³⁺ ions. The Concentration quenching effect was explained from Bless relation and Dexter energy transfer theory between activator ions. Decay curves of the prepared phosphors show bi-exponential behavior. The up-conversion emission spectra were recorded under 980 nm laser excitation. The optical transition in the UV–Vis range was investigated based on the diffuse reflectance spectra, and the results match well with the excitation spectra. The photometric study informs the color and the quality of the emitted light. The outcomes produce a new synthesis method to prepare La2Zr2O7:Pr³⁺ nano phosphor that can be an excellent candidate for solid-state lighting and UV/NIR to the visible converter in solar cell applications. Temperature-dependent long persistent luminescence properties and trap distributions in Sr2MgAl22O36: Eu2+	https://www.researchgate.net/publication/345894129_Photo_and_Mechano-luminescence_properties_of_novel_Zn_1-x_Eu_x_Al_2-y_Ba_y_O_4_advance_blue_phosphor
Griffith's Primary Valuation Records, Co. Antrim Richard Griffith, a geologist based in Dublin, became Boundary Commissioner in 1825 and Commissioner of Valuation in 1827. The results of his great survey, the Primary Valuation of Ireland, were published between 1847 and 1864. Griffith’s Primary Valuation Records, Co. Antrim From the 1820s to the 1840s a complex process of reform attempted to standardise the basis of local taxation in Ireland. The first steps were to map and fix administrative boundaries through the Ordnance Survey and the associated Boundary Commission. The next step was to assess the productive capacity of all property in the country in a thoroughly uniform way. Richard Griffith, a geologist based in Dublin, became Boundary Commissioner in 1825 and Commissioner of Valuation in 1827. The results of his great survey, the Primary Valuation of Ireland, were published between 1847 and 1864. The valuation is arranged by county, barony, Poor Law Union, civil parish and townland, and it lists every landholder and every householder in Ireland. Occupier_sur_name Immediate_lessor Townland County Coburn Mary Bolton, John Ballydonaghy Antrim Johnston Rose Anne Bolton, John Ballydonaghy Antrim Lavery Samuel Bolton, John Ballydonaghy Antrim M'Larnon Mary Bolton, John Ballydonaghy Antrim Smith William Browne, James Ballydonaghy Antrim Mallen Henry Bullock, William Ballydonaghy Antrim Lynn Charles Burke, Anne Ballysessy Antrim Kelly Mary Campbell, Jane Main street Antrim Magill Robert Campbell, Jane Main street Antrim Bell Andrew Charters, James Gorrana Antrim M'Keone Patrick Charters, James Gorrana Antrim Stewart Robert Charters, James Gorrana Antrim Adair William Coey Edward Sir Townparks Antrim Adrain Jacob Coey Edward Sir Ballycraigy Antrim Agnew William Coey Edward Sir Townparks Antrim Alexander James Coey Edward Sir Townparks Antrim Alexander Samuel Coey Edward Sir Antiville Antrim Alexander Samuel Coey Edward Sir Blackcave, South Antrim Allen Mary Coey Edward Sir Ballycraigy Antrim Allen Mary Coey Edward Sir Blackcave, North Antrim Atkinson Myles Coey Edward Sir Townparks Antrim Boyd Joseph Coey Edward Sir Antiville Antrim Boyd Joseph Coey Edward Sir Ballycraigy Antrim Boyd Josh. Thompson Coey Edward Sir Townparks Antrim Boyd Samuel & James Coey Edward Sir Ballycraigy Antrim Bruce William Coey Edward Sir Townparks Antrim Buchanan Alexander Coey Edward Sir Antiville Antrim Buchanan Alexander Coey Edward Sir Townparks Antrim Cairns Denis Coey Edward Sir Townparks Antrim Campbell James Coey Edward Sir Blackcave, South Antrim Carmichael James Coey Edward Sir Ballyloran Antrim ???? Archibald Coey Edward Sir Townparks Antrim ???? Margaret Anne Coey Edward Sir Ballyloran Antrim ???? Thomas Coey Edward Sir Townparks Antrim Coach James Coey Edward Sir Blackcave, South Antrim Couch James Coey Edward Sir Townparks Antrim Crawford John Coey Edward Sir Antiville Antrim Crawford John Coey Edward Sir Townparks Antrim Darcus Solomon Coey Edward Sir Townparks Antrim Dugan James Coey Edward Sir Townparks Antrim Ferguson David Coey Edward Sir Townparks Antrim Ferris Charles Coey Edward Sir Antiville Antrim Ferris Charles Coey Edward Sir Townparks Antrim Fitzsimmons Matthew Coey Edward Sir Townparks Antrim Fleming Malcolm Coey Edward Sir Townparks Antrim Gibson Robert Coey Edward Sir Townparks Antrim Hamilton Alexander Coey Edward Sir Townparks Antrim Hunter Anne Coey Edward Sir Ballycraigy Antrim Hunter James Coey Edward Sir Townparks Antrim Hutton James Coey Edward Sir Townparks Antrim Kilpatrick Thomas Coey Edward Sir Blackcave, South Antrim Kirkpatrick Alexander Coey Edward Sir Blackcave, South Antrim Lorimer Eliza Coey Edward Sir Townparks Antrim Magill Arthur Coey Edward Sir Antiville Antrim Maninch Robert Coey Edward Sir Ballycraigy Antrim Martin Charles Coey Edward Sir Antiville Antrim M'Clarkin Robert Coey Edward Sir Townparks Antrim M'Cormack Daniel Coey Edward Sir Ballyloran Antrim M'Dowell Alexander Coey Edward Sir Antiville Antrim M'Fall Archibald, jun Coey Edward Sir Ballyloran Antrim M'Fall?? Archibald, sen. Coey Edward Sir Ballyloran Antrim M'Garel?? Helena Coey Edward Sir Townparks Antrim M'Garrell William Coey Edward Sir Townparks Antrim Miller Isaac Coey Edward Sir Townparks Antrim M'Kinty Sarah Coey Edward Sir Blackcave, South Antrim M'Master James Coey Edward Sir Townparks Antrim M'Neill Henry Coey Edward Sir Townparks Antrim M'Neill John Coey Edward Sir Blackcave, North Antrim M'Neill John x 2 Coey Edward Sir Townparks Antrim M'Neill Joseph Coey Edward Sir Blackcave, North Antrim Molyneaux H. M., Rev. Coey Edward Sir Townparks Antrim Molyneaux Samuel Coey Edward Sir Townparks Antrim Molyneaux Samuel, Rev. Coey Edward Sir Ballyloran Antrim Moone William Coey Edward Sir Townparks Antrim Moore Alexander Coey Edward Sir Blackcave, South Antrim Paden Ellen Lorimer Coey Edward Sir Ballycraigy Antrim Rainey Thomas Coey Edward Sir Antiville Antrim Ritchie John Coey Edward Sir Blackcave, South Antrim Ross James Coey Edward Sir Townparks Antrim Smyth John Coey Edward Sir Townparks Antrim Snoddy Samuel Coey Edward Sir Antiville Antrim Wilson Hugh Coey Edward Sir Townparks Antrim Workman Abraham Coey Edward Sir Blackcave, South Antrim Workman Jane Coey Edward Sir Ballycraigy Antrim Workman William Coey Edward Sir Ballyloran Antrim M'Clooney William Cousins, Margaret Ballytromery Antrim Moore Archibald Dixon Thomas Townparks Antrim M'Keever William Donaldson, Samuel Ballytromery Antrim Carothers Samuel Douglas, George Gorrana Antrim Clifford George Douglas, George Gorrana Antrim Hall John Douglas, George Gorrana Antrim Hall Stephen Douglas, George Gorrana Antrim Nelson Robert Douglas, George Gorrana Antrim Young William Douglas, George Gorrana Antrim Coey , Sir Edward Earl of Antrim Blackcave, South Antrim Cardwell Jane Ferris, Arthur Ballysessy Antrim Elliott John Ferris, Arthur Ballysessy Antrim Smith James Ferris, Arthur Ballysessy Antrim Bullock John Ferris, George Ballysessy Antrim M'Carr William Ferris, George Ballysessy Antrim My tables are sorted alphabetically by Occupiers’ name – any Immediate Lessor may be listed numerous times throughout a parish. Immediate Lessor names are not sorted alphebetically In those instances when a person is listed twice in the same townland and the Immediate lessor was the same for each entry I have the occupiers name once and indicated how many times that name occurs in that townland beside it. I have excluded the name of Immediate Lessors of Vacant properties when that person was already listed as being an Immediate Lessor in the same townland. The names of Immediate Lessors of vacant properties are included if that person was not listed as an Immediate Occupier previously in that townland. In those instances when a person is listed twice in the same townland and the Immediate lessor was the same for each entry I have the occupiers name once and indicated how many times that name occurs in that townland beside it. I have excluded the name of Immediate Lessors of Vacant properties when that person was already listed as being an Immediate Lessor in the same townland. The names of Immediate Lessors of vacant properties are included if that person was not listed as an Immediate Occupier previously in that townland. The Immediate Lessor is the person who owns or leases the property from the Landholder. The Occupier is the person who leases the land from the Immediate Lessor. The Occupier, in turn, may sub-let part of the property that they are listed as being the Occupier of, in which case, their name is then listed in the Immediate Lessor column. You need to see the original pages of the Griffiths Valuation which lists the type of property being leased.	https://www.from-ireland.net/griffiths-primary-valuation/antrim/?http%3A%2F%2Fhttp%253A%252F%252Fhttp%25253A%25252F%25252Fhttp%2525253A%2525252F%2525252Fhttp%252525253A%252525252F%252525252Fhttp%25252525253A%25252525252F%25252525252Fhttp%2525252525253A%2525252525252F%2525252525252Fpg=1&pg=2pg=4pg=3pg=3pg=4pg=3pg=3
Polyamines in Goat's Colostrum and Milk Download Citation \| Polyamines in Goat's Colostrum and Milk \| The concentration and secretion of putrescine (PTR), spermidine (SPD), and spermine (SPM) was examined in colostrum and milk of 60 dairy goats... \| Find, read and cite all the research you need on ResearchGate Polyamines in Goat's Colostrum and Milk October 1997 Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 118(1):45-52 DOI: 10.1016/S0305-0491(97)00018-7 Authors: T Motyl Request full-text Abstract The concentration and secretion of putrescine (PTR), spermidine (SPD), and spermine (SPM) was examined in colostrum and milk of 60 dairy goats (Polish White and German Brown) during 90 days of lactation. It has been found that milk polyamine pattern is related to breed, age, offspring number, lactation period, milking time, and individual goat-to-goat variations. The mean level of PTR and SPD was significantly higher, whereas SPM level was significantly lower in German Brown than Polish White goats. These differences were maintained during whole observation period (90 days after parturition). Elder goats, bearing two and three kids, secreted colostrum with a higher concentration of PTR and a lower concentration of SPD than young goats having one kid. There was a highly significant positive relationship between the age or litter total weight and PTR concentration in colostrum of Polish White goats. In contrast to PTR, the SPD level was negatively correlated with the age or litter total weight. The daily secretion of PTR, SPD, and SPM progressively increased from the 1st to 90th day of lactation. It was dependent on the increase of milk secretion rate, which could be illustrated by the highly significant correlation between daily milk yield and secretion of SPD in milk of German Brown and Polish White goats. The concentration of SPD in milk sampled in the evening was usually higher than in that from morning milking, however, significant only in the case of German Brown goats. In general, goat's colostrum and milk are rich in polyamines, which the total concentration is the highest when compared to milk of other mammals examined so far (e.g., human, rat, sow, and cow). ... The 60 percent of students consumed junk food on a daily basis and 70 percent of them were not aware of nutritional content of food consumed by them. This study emphasises education on health hazards of junk food among adolescents [14] . ... ... The findings were contradicted with the Social and Health Survey of Children and Adolescents examined the association between family break-up and psychosocial maladjustment in adolescents, which showed changes in family structure, is associated with an increase in psychosocial maladjustment among adolescents [14] . ... ... Polyamines play an important role in maturation of the GIT enzymes, cell function [12] and reduce the incidence of food allergy in infants [13]. The concentration of polyamines in goat milk is highest in goat milk compared to cow and human milk [14] . It was found that five times more goat milk is required to trigger an adverse reaction than the cow milk [15]. ... Nephrotic syndrome: case report ... Cow milk and dairy products have lower polyamine contents than human milk does because of the high rate of polyamine degradation in the former by highly active enzymes such as diamine oxidase or polyamine oxidase (Löser, 2000). Studies on polyamines in milk from small ruminants are very limited (Ploszaj et al., 1997; Prosser et al., 2008;Galitsopoulou et al., 2015). Comparing colostrum and milk from two goat breeds (German Brown and Polish White) over 90 days of lactation, Ploszaj et al. (1997) indicated that goat colostrum and milk are rich in polyamines and that their total concentration is higher than that observed in milk from other mammals. ... ... Studies on polyamines in milk from small ruminants are very limited (Ploszaj et al., 1997;Prosser et al., 2008;Galitsopoulou et al., 2015). Comparing colostrum and milk from two goat breeds (German Brown and Polish White) over 90 days of lactation, Ploszaj et al. (1997) indicated that goat colostrum and milk are rich in polyamines and that their total concentration is higher than that observed in milk from other mammals. On the whole, the SPM concentration (3.18 μmol/L in German Brown goats and 3.80 μmol/L in Polish White goats) was similar to that determined in human milk, whereas the SPD content (39.67 μmol/L in German Brown goats and 26.00 μmol/L in Polish White goats) was higher than that in humans; the PTR level (6.00 μmol/L in German Brown and 5.12 μmol/L in Polish White) was highest among other mammals (human, cow, rat and sow). ... ... On the whole, the SPM concentration (3.18 μmol/L in German Brown goats and 3.80 μmol/L in Polish White goats) was similar to that determined in human milk, whereas the SPD content (39.67 μmol/L in German Brown goats and 26.00 μmol/L in Polish White goats) was higher than that in humans; the PTR level (6.00 μmol/L in German Brown and 5.12 μmol/L in Polish White) was highest among other mammals (human, cow, rat and sow). The results presented by Ploszaj et al. (1997) highlight a relation among polyamine patterns, goat age, offspring number, lactation period, milking time and individual variation. In the lactation stage, the SPM and SPD milk concentrations remained relatively stable over 90 days of lactation, whereas PTR decreased during the first month of lactation. ... Oligosaccharides, polyamines and sphingolipids in ruminant milk Article Jan 2018 SMALL RUMINANT RES Daniela Giorgio Adriana Di Trana Claps Salvatore As a result of the widespread applications and the use of milk and milk products in human nutrition, intense scientific interest has focused on identifying the components in milk that may be relevant to improving human health. Milk contains a heterogeneous mixture of secretory compounds with a wide variety of chemical and functional activities. Among these compounds, many indigenous minor bioactive compounds are present. This review provides an overview of the available knowledge on the polyamines, oligosaccharides and sphingolipids occurring in goat and sheep milk compared to cow milk. It has been highlighted that goat milk is richer in polyamines than is milk from other mammals and that the former represents a very appealing source of human-like oligosaccharides. Furthermore, it is interesting to note the effect that breed and polymorphism at the CSN1S1 locus exerts on the polar lipid content and oligosaccharides profile. Goat and sheep milk production plays an important role in the nutrition and economy of many countries around the world, especially in the Mediterranean area. Information on the bioactive properties of goat and sheep milk is an important tool for their use as a nutritional source for infants, in medicinal foods and for developing new markets. ... BAs were extracted from the milk samples by acid treatment [15] . Briefly, 500 μL of milk were precipitated with 100 μL of trichloroacetic acid (12% v/v) for 1 h. ... ... Spearman correlation coefficients between different BAs were also determined. Calculations were made using either SPSS v. 15 ... ... This agrees with that reported by other authors [18,19]. Factors such as the length of time a mother has been lactating, and her age, are known to affect milk PA concentrations [15] . This might also explain the different PA concentrations reported in previous studies [9,18,19]. ... Mastitis Modifies the Biogenic Amines Profile in Human Milk, with Significant Changes in the Presence of Histamine, Putrescine and Spermine Full-text available Sep 2016 PLOS ONE Begoña Redruello Biogenic amines (BAs) are low molecular weight nitrogenous organic compounds with different biological activities. Putrescine, spermidine and spermine are essential for the development of the gut and immune system of newborns, and are all found in human milk. Little is known, however, about the role of histamine, tyramine or cadaverine in breast milk. Nor is it known whether mastitis alters the BA composition of milk. The BA profile of human milk, and the influence of mastitis on BA concentrations, were therefore investigated. Putrescine, spermidine and spermine were the main BAs detected. In mastitis-affected milk, the concentrations of putrescine, spermine and histamine were higher. ... To the best of our knowledge, there are no detailed studies on ovine milk polyamine contents (Michaelidou, 2008). Studies on polyamines in goat's milk are also limited (Prosser, Mclaren, Frost, Agnew, & Lowry, 2008; Płoszaj, Ryniewicz, & Motyl, 1997) . Płoszaj et al. (1997) reported higher values for individual polyamines, compared to milk of other mammals (e.g., human, rat, sow, and cow). ... ... Studies on polyamines in goat's milk are also limited (Prosser, Mclaren, Frost, Agnew, & Lowry, 2008;Płoszaj, Ryniewicz, & Motyl, 1997). Płoszaj et al. (1997) reported higher values for individual polyamines, compared to milk of other mammals (e.g., human, rat, sow, and cow). Prosser et al. (2008) also demonstrated that goat whole milk powder and goat-based infant and follow-on formulas were richer in polyamine content than their cow milk counterparts. ... ... Other plausible explanations may include the circadian rhythm of polyamine secretion and the age of the animals. The latter is most probably related to differences in hormonal levels between younger and older animals that may, in turn, alter the type of signalling on the mammary gland during lactation (Płoszaj et al., 1997) . According to La Torre et al. (2010), differences in the concentration of these bioactive compounds are difficult to interpret due to an inherent multifactorial variability. ... Polyamine profile in ovine and caprine colostrum and milk FOOD CHEM Alexandra Michaelidou George Menexes Efstathios Alichanidis The objective of this study was to monitor the post-partum variation of polyamine content, in ovine and caprine milk, from indigenous Greek breeds. Twenty samples of ewe and 20 samples of goat colostrum and milk were collected at the 1st, 2nd, 3rd, 4th, 5th and 15th day post-partum. Putrescine, spermidine and spermine were measured as dansylated derivatives by high-performance liquid chromatography. Putrescine was the least concentrated of these substances in both milk types. Spermidine was the prevailing polyamine in caprine samples, reaching levels up to 4.41 μmol/l on the 3rd day post-partum. In ovine milk, the profile of the mean concentrations showed greater levels of spermine than spermidine, except for the 5th day post-partum. These data suggest that goat colostrum and ewe milk (15th day) could be considered as good natural sources for these bioactive growth factors, and may become useful raw materials for designing tailored dairy products for specific population groups. ... Daily oscillation in the levels of physiological variables has been described for a multitude of variables, including locomotor activity, body temperature, heart rate, blood pressure, hormonal secretion, and urinary excretion (Dunlap et al., 2004;Refinetti, 2006), and it is to be expected that milk composition will also exhibit daily variation. Indeed, daily variation in milk composition has been documented in humans (Lubetzky et al., 2006;Stafford et al., 1994) as well as in ruminants (Nielsen et al., 2003; Płoszaj et al., 1997) . ... ... Except for a gradual reduction in lipid content, there was no significant change in rhythmic parameters throughout the lactation period from 1 to 7 months postpartum. These results confirm and expand the results of previous studies documenting daily variation of milk composition in humans (Lubetzky et al., 2006;Stafford et al., 1994) and ruminants (Nielsen et al., 2003; Płoszaj et al., 1997) . ... Daily rhythmicity in nutrient content of asinine milk Asinine (donkey) milk has been proposed as an alternative to bovine (cow) milk for consumption by human infants who are allergic to bovine milk proteins. To expand the current knowledge of asinine lactation, we investigated daily rhythmicity in the concentration of major constituents of asinine milk. We observed no daily rhythmicity in somatic cell count and pH but observed robust rhythmicity in lipid, lactose, and protein content (robustness greater than 70%, p < 0.0 1). Lipid and lactose content peaked during the night, whereas protein content peaked during the day. Lipid content of asinine milk was lower than that of human milk, and asinine milk resembled human milk better than bovine milk regarding sugar and protein content. ... Furthermore , the polyamines putrescine, spermine and spermidine are present in all cells at low levels, and play fundamental roles in DNA, RNA and protein synthesis (Löser, 2000). Motyl et al. (1995) and Ploszaj et al. (1997) investigated polyamine levels in milk from goats, sows and cows. Levels peak in the colostral phase and early lactation. ... ... We investigated the effects of biogenic amines on two types of bovine epithelial cell cultures to throw light on the effect of these amines on mammary gland. Most of the previous studies conducted in vivo have focused on milk composition in polyamines in relation to their role in the gut development and maturation of the newborns (Motyl et al., 1995; Ploszaj et al., 1997 ). The in vitro studies in mammary glands in mice (Kano and Oka, 1976 ; Oka and Per- ry, 1983) indeed investigated the transport and the metabolism of these compounds. ... Effects of putrescine, cadaverine, spermine, spermidine and β-phenylethylamine on cultured bovine mammary epithelial cells Antonella Baldi Federica Cheli Stig Purup A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermidine and β-phenylethylamine on mammary epithelial cells. Each biogenic amine was diluted in several concentrations (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures. In BME-UV1, spermine significantly inhibited cell proliferation (P<0.001), while the other amines inhibited at higher concentrations (50mM). In primary bovine organoids, β−phenylethylamine significantly (P<0.001) inhibited cell proliferation at 4 mM. Organoids cultured in the presence of all amines, except β-phenylethylamine, had stellate projections indicating intense cell proliferation. Proliferation of mammary epithelial cells was stimulated at low concentrations, while at high concentrations it was inhibited. Our results suggested that the effects of each compound on mammary epithelial cells could be related to the compound itself and not to mediating by the bovine amino oxidase, responsible of the formation of toxic metabolites. ... Allergy to cow milk is a common problem with a prevalence rate of 2.5% among children during the (Prosser et al., 2008) Immune maturation of milk-fed offspring, mediation of energy metabolism, signal transduction and general regulation of cell growth, lipoprotein metabolism, enhancement of high-density lipoprotein (HDL) plasma concentration, synthesis of apolipoprotein (Apo) A1 and Apo A1 V in pre-term infants, and upregulation of longchain polyunsaturated fatty acid synthesis in human neonates (Schaller et al., 2007) Taurine 2 0 -4 0 f o l d s h i g h e r t h a n c o w m i l k (Silanikove et al., 2010) Bile salt formation, osmoregulation, antioxidation, calcium transport and functioning of the central nervous system (Silanikove et al., 2010) Regulate blood pressure and alleviate other cardiovascular ailments (Militante and Lombardini, 2002) Polyamines Rich in goat colostrum and milk compared to other mammals (Ploszaj et al., 1997) Optimise growth, gastrointestinal tract (GIT) cell function, maturation of GIT enzymes (Silanikove et al., 2010) and reduce the incidence of food allergy in infants (Dandrifosse et al., 2000) Oligosaccharides 4-5 times higher than cow milk and 10 times higher than sheep milk Antiadhesive, anti-microbial, immune modulators; intestinal epithelial cell response modulation, nutrient for neonatal brain development and growth of desired gut microflora (Lima et al., 2018) first three years of life. Replacement of cow milk with goat milk resolved between 30 and 40% of the cases. ... Production, processing and marketing of goat milk in India: Reality and scope Full-text available Jan 2021 Arun K. Verma ... In comparison to the cow milk the peculiarities of the goat milk concern many aspects: goat milk has smaller fat globules, higher proportions of medium-chain fatty acids, smaller casein micelles, low levels of ␣ s1 -casein and higher proportion of ␤-casein (Clark and Sherbon, 2000), higher level of some essential amino acids (Posati and Orr, 1976), higher level of free amino acid taurine (Mehaia and Al-Kanhal, 1992), higher concentration of some whey proteins, higher content of calcium, phosphorous and potassium, similar content of vitamin, a complex array of nucleotides (Prosser et al., 2008), a good source of polyamides (Ploszaj et al., 1997) and lower lactose content. ... Factor affecting the 3′ sialyllactose, 6′ sialyllactose and disialyllactose content in caprine colostrum and milk: Breed and parity SMALL RUMINANT RES Claps Salvatore A.R. Caputo ... This observation led these authors to suggest that dietary polyamines can playa role in the intestinal maturation at weaning. Otherwise, it has been shown that milk from sow (Kelly et al., 1991;Motyl et al., 1995), cow (Motyl et al., 1995) and goat (Ploszaj et al., 1997) contain significant amounts of polyamines. ... Chapter 4 Maturation of intestinal digestive and immune systems by food polyamines Olivier Peulen P Deloyer G Dandrifosse Maturation of the intestine occurs during the first few postnatal weeks in many vertebrates. This maturation allows the pups to adapt to the drastic change from milk to solid food. The enzymes located in the mucosal brush border adapt to solid food: lactase specific activity decreases, in contrast to maltase and sucrase activities that increases. Polyamines are involved in gut maturation in rats. Spermine administration leads to an increase of plasma adrenocorticotrophic hormone (ACTH) and corticosterone, and bilateral adrenalectomy drastically reduces the spermine effect on the small intestine. This chapter describes the phenomena observed in the small intestine after ingestion of spermine and compares them to the natural postnatal maturation occurring at weaning. It proposes a mechanism of spermine action in small intestine maturation, describes the spermine-induced maturation of the spleen, and explores the impact of this substance in human health. Spermine ingested by suckling rats induces the secretion of soluble factors in the circulation. These factors are responsible for hypothalamo-hypophyso-adrenal axis activation. Spermine administration provokes cytokine secretion in plasma, cytokine injection induces a precocious maturation of the small intestine, and spermine administration is responsible for immune system maturation. Polyamines are involved in gut maturation and the polyamine concentration in the intestinal mucosa increases at weaning. ... additional Nu (Prosser et al., 2008). Polyamines, another component of the non-protein fraction of milk was shown to be important for optimal growth, gastro intestinal tract (GIT) cell function, maturation of GIT enzymes (Pegg and McCann, 1982) and have been implicated in reducing the incidence of food allergy in infants (Dandrifosse et al., 2000). Ploszaj et al. (1997) found that goat colostrum and milk are rich in polyamines, highest compared to milk of other mammals (e.g., human, rat, sow, and cow). Prosser et al. (2008) found that goat milk and goat-based infant and follow-on formulas were marginally richer in polyamine content than cow milk or cow milk-based formulas; though in both species the le ... Recent advances in exploiting goat's milk: Quality, safety and production aspects SMALL RUMINANT RES MERIN C PROSSER CG Goat milk production is a dynamic and growing industry that is fundamental to the wellbeing of hundreds of millions of people worldwide and is an important part of the economy in many countries. The aim of the present review is to provide an integrated and critical analysis of the major aspects in this field to highlight unexploited nutritional potential of goat milk and the need for improvements, particularly in food safety. First, it should be kept in mind that goat milk like cow milk delivers many nutrients with relatively low energy content, and is relevant to the health of consumers throughout the life cycle. In addition, the review presents data suggesting that goat milk possesses many advantages over cow milk, for use as a nutritional source for infants and children and as a medicinal food. Furthermore, goats, by consuming large amounts of natural browsing plants all year around, are a potentially overlooked “treasure trove”, with respect to health promoting components. The survey suggests that total bacterial count that is currently used as the major quality measure to prevent pathogen-related food toxicity is not sufficiently effective. The proposal is to include somatic cell count as a routine criterion to qualify the hygienic status of goat milk in terms of the relevant physiology and biochemistry. The paper presents a novel mechanism controlling milk secretion, and demonstrates the use of this knowledge in making decisions for two major managerial tasks that farmers face, namely milking frequency that dictates to a large extent the milk yield and workload on the farm, and helping to deal with subclinical mastitis that is the single major cause for economical losses in dairy farms worldwide. ... creased in the villus and crypt cells by spermine Polyamine concentrations of the milk were also ingestion. All these observations suggest that oral estimated in the cow (Sanguansermsri et al., 1974; ingestion of spermine induces complete intestinalMotyl et al., 1995), sow (Kelly et al., 1991;Motyl et al., 1995), goat ( Poszaj et al., 1997 ) and human which was never observed after weaning. Neverthefemale (Sanguansermsri et al., 1974;Pollack et al., less, irreversibility of normal intestinal maturation 1992;Romain et al., 1992;Buts et al., 1995). ... Intestinal maturation induced by spermine in young animals Livest Prod Sci Olivier Peulen P Deloyer Ch. Grandfils G Dandrifosse In suckling rats, it has been established that oral administration of spermine, a dietary polyamine, at appropriate doses, induces all the modifications in the digestive tract that occur at weaning, namely, (a) in the intestine: variations in the specific activities of disaccharidases and peptidases, gene expression, the level of receptors to polymeric immunoglobulins (RPIs), tissue histology, and the intestinal permeability to macromolecules; (b) maturation of the intestinal immunological system, an observation confirmed in mice; (c) an increase in the specific activity of enzymes contained in the pancreas; (d) a change in the growth rate and biochemical properties of the liver, e.g., in the synthesis of RPIs. The mechanism of spermine action is partly understood. In the intestine, two phases of events have been recorded. The first phase consists of the desquamation of the epithelium resulting from an activation of apoptosis, probably induced by the entrance of spermine into the enterocytes. The second phase concerns a hormonal cascade in which adrenocorticotrophic hormone, cytokines, bombesin and corticosterone intervene. A general hypothesis taking into account all the results obtained until now is presented as well as other observations recorded on the effect of exogenous spermine on the intestinal maturation of vertebrates other than the rat. ... Besides their role in the normal renewal of the adult intestinal epithelium, PAs seem also to be required for enterocyte maturation. For instance, PAs found in the milk Pollack et al. 1992;Motyl et al. 1995; Ploszaj et al. 1997) could exert a stimulatory effect on both the postnatal growth and the maturation of the intestinal mucosa in suckling animals and human newborns. Indeed, the oral administration of SPM (a natural component of human and rat milk) to suckling rats is correlated with the precocious appearance of some characteristics typical of the mature intestine (Dufour et al. 1988;Dandrifosse et al. 1992;Buts et al. 1993;W ery & Dandrifosse, 1993;Dorhout et al. 1997). ... Pancreatic Exocrine Secretions as a Source of Luminal Polyamines in Pigs EXP PHYSIOL Suzanne Loret Philippe Brolet Stefan G. Pierzynowski Guy Dandrifosse The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA-induced growth and differentiation of porcine intestinal epithelium. ... High dietary intake of polyamine has been implicated in reducing the incidence of food allergy in infants (Peulen et al. 1998;Duchen & Thorell 1999, Dandrifosse et al. 2000. The present study found that the concentrations of polyamines in goat milk powders and infant and follow-on formulae are slightly higher than cow milk or cow-based formulae, which confirms earlier data for raw milk (Ploszaj et al. 1997) . However, the levels present in goat or cow infant formula are much lower than levels found in human milk (Romain et al. 1992) and represent only a minor component of the nonprotein nitrogen fraction of these formulae. ... Composition on non-protein nitrogen fraction of goat whole milk powder and goat milk-based infant and follow-on formulae Deborah Frost Dianne Lowry The non-protein nitrogen fraction of goat whole milk powder and of infant and follow-on formulae made from goat milk was characterized and compared with cow milk powder and formulae. Goat milk infant formula contained 10% non-protein nitrogen, expressed as a proportion of total nitrogen, compared with 7.1% for cow milk formula. Goat follow-on formula contained 9.3% and cow 7.4% non-protein nitrogen. Urea, at 30%, was quantitatively the most abundant component of the non-protein nitrogen fraction of goat milk and formulae, followed by free amino acids at 7%. Taurine, glycine and glutamic acid were the most abundant free amino acids in goat milk powders. Goat milk infant formula contained 4 mg/100 ml total nucleotide monophosphates, all derived from the goat milk itself. Goat milk has a very different profile of the non-protein nitrogen fraction to cow milk, with several constituents such as nucleotides at concentrations approaching those in human breast milk. Exploring potential of goat based dairy farming in India and way forward Manoj Kumar Singh S K Singh The aim of this review is to highlight the significance of goat milk along with potential and prospects of dairy goat development in the country. India occupies the first position in goat milk production in the world. In the last few years, commercial dairy goat production in India gained momentum due to spread of knowledge about therapeutic, nutraceutical and medicinal benefits of goat milk and its product, and their export potential. India possesses vast caprine resources with 37 goat breeds distributed in different bio-climates with varied nutritive value, however, some goat breeds native to north and northwestern region namely Beetal, Jamunapari, Jakhrana, Surti and Zalawadi are considered as Indian dairy breed with 150 to 500 litre milk yields. The reported milk yield of Indian dairy goat is far below their potential, since they are primarily raised for mutton and also due to energy-deficient diet. Attempts so far made were scarce and limited for milk improvement of dairy goats and in creating infrastructure for goat milk processing and marketing. Use of potential sire/semen and infrastructural support for a secured market for goat milk and products is necessary to enhance dairy goat productivity and profitability. To cater the demand for goat milk in southern, eastern and hilly regions, suitable dairy breeds need to be developed along with increasing the genetic potential of existing breeds. Development of the goat dairy sector will require focused efforts to encourage entrepreneurship to set as many as possible commercial dairy farms by involving private sector through appropriate policy support and incentives. Goat Milk – Chemistry and Nutrition Interest in dairy goats and goat milk products is a part of the recent trend in health food demand and consumption in developed countries as well as a renewed interest in goat milk as a substitute for those who suffer from allergies or intolerance against cow milk. The major nutrient composition, flavor, and appearance of goat milk resemble cow milk, but goat milk has its unique chemical, biochemical, physical, and nutritional characteristics compared to cow and other species milk. Goat milk contains slightly less total casein, but higher non-protein nitrogen than the cow counterpart. The most remarkable difference in basic composition between goat (or cow) milk and human milk exists in protein and ash contents. The α-lactalbumins play an important role in milk biochemistry, since they are part of the lactose-synthetase enzyme involved in synthesis of lactose. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells Eleonora Fusi Antonella Baldi Federica Cheli Stig Purup A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermicline and beta-phenylethylamine on mammary epithelial cells. Each biogenic amine was diluted in several concentrations (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures. In BME-UV1, spermine significantly inhibited cell proliferation (P<0.001), while the other amines inhibited at higher concentrations (50mM). In primary bovine organoids, beta-phenylethylamine significantly (P<0.001) inhibited cell proliferation at 4 mM. Organoids cultured in the presence of all amines, except beta-phenylethylamine, had stellate projections indicating intense cell proliferation. Proliferation of mammary epithelial cells was stimulated at low concentrations, while at high concentrations it was inhibited, Our results suggested that the effects of each compound on mammary epithelial cells could be related to the compound itself and not to mediating by the bovine amino oxidase, responsible of the formation of toxic metabolites. Leite fermentado: Potencial alimento funcional Article Marion P Costa Rodrigo Moreira Goat's milk for infants and children? Pediatr Pol In our study we reviewed the literature about the qualitative and quantitative composition and nutritive value of goat's milk compared with cow's milk. We focused on literature data about the use of goat's milk in children with cow's milk allergy. Polyamines and cancer: Scientific basis and therapeutic potentialities Polyamines are universal and mandatory molecular components for any living cell. They are ubiquitous in the cell and in the organism and are essential for vital functions and growth. Their numerous potential targets as well as their aptitude to modify these targets' physical and/or biological properties explain their wide spectrum of activity. Polyamine metabolism as such is an attractive target for anticancer therapeutic intervention. But, blocking or deregulating their metabolism is insufficient as hyper-proliferative cells have access to important exogenous polyamines pools mainly provided by food and to a lesser extent, gut bacteria. The potential of polyamine metabolism inhibitors associated with polyamine-deficient food and gut decontamination has been assessed in pre-clinical studies demonstrating in vivo very significant inhibition of tumor growth and metastatic spread. Furthermore, polyamine depletion enhances lowdose chemotherapy. Preliminary studies on patients with hormone-refractory metastatic prostate cancer showed that low polyamine-containing diet is applicable in these patients, well-tolerated with a good compliance, and allows improvement of quality of life. Easily prescribed in elderly patients, this novel, non toxic nutritional approach is actually investigated in phase II clinical trials. View Terapijska i zdravstvena svojstva kozjeg mlijeka/ Nutritional and therapeutic properties of goat’s milk Production of goat milk and its consumption in the world is increasing, and so is the population of goats which increases more than the population of other dairy animals. This is particularly true in countries where goat milk is reflection of the traditional production. Goat’s milk, in addition to the high nutritional value (better digestibility, hypoallergenic, higher buffering capacity, higher pH value) is characterized by therapeutic characteristics important for human health. The preference of goat’s milk over cow’s milk is also a higher selenium content, which is essential for the activity of the enzyme glutathione peroxidase, also significant in the prevention of cancer and cardiovascular diseases. Goat’s milk in comparison with cow’s, contains more fatty acids, which have beneficial effects on human health, especially the cardiovascular system. Goat’s milk proteins are more digestible than cow’s milk proteins, also an absorption of amino acids are more efficient. The sensitivity of people to α-lactalbumin and β-lactoglobulin of cow’s milk is negligible after replacing cow’s milk with goat’s milk. The objective of this paper is to specify benefits of goat’s milk in regard to cow’s and highlight its therapeutic and nutritional values. Leite fermentado: potencial alimento funcional / Fermented milk: potential functional food RESUMO O leite fermentado apresenta um elevado potencial no desenvolvimento de novos produtos, principalmente por estar relacionado com a saúde e com a praticidade no consumo. Recentemente, as indústrias de alimentos têm explorado essa matriz devido a suas características, que permite a viabilidade funcional dos ingredientes adicionados, a aceitabilidade dos produtos lácteos, e a relação que os consumidores fazem com o aspecto de saudabilidade. Os alimentos funcionais são todos os alimentos ou bebidas que, consumidos na alimentação diária, podem conferir benefícios fisiológicos específicos, devido à presença de compostos biologicamente ativos. Estes alimentos podem ser classificados de acordo com o alimento em si, ou conforme os componentes bioativos nele presentes como, por exemplo, os probióticos, os prebióticos e os compostos funcionais. Os probióticos são micro-organismos vivos, que quando ingeridos em quantidades adequadas conferem benefícios à saúde do hospedeiro. Enquanto os prebióticos são carboidratos seletivamente fermentáveis que permitem modificações específicas na diversidade e/ou na atividade da microbiota gastrointestinal, resultando em benefícios ao bem estar e à saúde do hospedeiro. Neste mesmo contexto de aporte de componentes salutares através da ingestão de alimentos estão inseridos os compostos funcionais, como o ácido linoleico conjugado, o folato, a colina e as poliaminas. Contudo, deve-se salientar que para a obtenção destes benefícios, o consumidor deve ingerir uma dose mínima de consumo diário destes produtos funcionais, associado a um hábito de vida saudável, que inclui a prática de exercícios regulares e uma alimentação balanceada. View Factors influencing nutritional and health profile of milk and milk products SMALL RUMINANT RES Milk has long been and will always be consumed as part of a healthy balanced diet as it contains an impressive array of nutrients and, therefore, plays an important role in assisting individuals to meet their nutrient requirements. Nowadays, that nutrition science moves beyond the study of essential nutrients, interspecies differences, considered as a symptom of evolutionary adaptation to different demands of neonates, could be of major biological importance. Research over the last twenty years has focused on bovine milk. Hence, sheep and goat milk still remain an appealing area of research. The possibility to develop easy digestible ovine and/or caprine milk products, for consumer groups with special needs, such as infants, athletes and the elderly is gathering momentum. Furthermore ovine and caprine milk products may carry specific health effects, when ingested as part of the daily diet; they may be convenient vehicles for peptides that reduce the activity of the angiotensin-converting enzyme, which is involved in vasoconstriction and therefore blood pressure. The presence of casein phosphopeptides may also enhance the physiological significance of ovine and caprine dairy products. Components of the non-protein-nitrogen fraction of milk that may be able to transmit biochemical messages, such as polyamines and nucleotides have also attracted particular scientific attention. Further exploration of ovine and caprine milk to understand its wholesomeness and the functions of intact milk is warranted. Additionally the possibility of using ovine or caprine milk components or colostral fractions for future product development needs further investigation. Polyamines in sow colostrum and milk at different stages of lactation Anim Sci Z. B. Cheng D.F. Li C.R. Ge The objective of this study was to determine the effect of stage of lactation on the concentration of polyamines (spermine, spermidine, and putrescine) in sow colostrum and milk. Ten, third parity sows (Landrace×Yorkshire) were sampled throughout a 28-day lactation. Litter size for all sows was standardized at 10 sucking piglets immediately after farrowing. Colostrum collection began at the birth of the first piglet (0 h), and colostrum was subsequently collected 6, 12, 18, and 24 h after farrowing. Sow milk was sampled on days 3, 7, 10, 14, 17, 21, 24 and 28 of lactation. Spermine concentration in sow colostrum was relatively constant for the first 12 h after farrowing and then rapidly increased to 10·26 μmol/l 24 h after farrowing (linear and quadratic effect; P<0·01). In contrast, the concentrations of spermidine and putrescine showed little change during the first 24 h with spermidine concentration in colostrum ranging from 3·76 to 4·54 μmol/l, and putrescine concentration ranging from 1·15 to 1·52 μmol/l. The highest concentration of spermine in sow milk was 18·82 μmol/l, observed on day 7, with levels gradually declining to 4·28 μmol/l by day 28 of lactation (linear and quadratic effect; P<0·01). In contrast, the concentration of spermidine increased during lactation ranging from a low of 4·32 μmol/l on day 7 to a high of 9·01 μmol/l on day 28 of lactation (linear effect P<0·01; quadratic effect P=0·03). Putrescine concentration ranged from 1·53 μmol/l on day 3 to 1·84 μmol/l on day 28 (linear effect P=0·07; quadratic effect P>0·05). The results of this study show sow colostrum and milk provide polyamines for sucking piglets before weaning, which could potentially modulate intestinal maturation. Optimization of the analytical extraction of polyamines from milk Article Oct 2011 Polyamines play an important role as growth promoters, in the maturation of the intestinal tract of infants and in the modulation of the immune response; consequently, the importance of polyamines in the diet of infants and adults is well established. However, information on the occurrence and levels of polyamines in cow's milk and milk from other species (including human milk) is contradictory. Furthermore, the methods used for the extraction of amines from these samples vary widely. Therefore, a method for the accurate analysis of amines in milk from different species and in milk products is needed. A sequential strategy of experimental designs was used to optimize the analytical extraction of polyamines from milk. The dependent variables that significantly affected the recoveries were screened through a Plackett-Burman design. Sulfosalicylic acid (SSA) provided better recoveries compared to trichloroacetic acid. Centrifugation time and speed during extraction were independent variables. The Central Composite Rotational Design used to optimize the dependent variables indicated that the optimal conditions for the extraction of polyamines were 40s vortexing, four successive extractions, and an SSA concentration of 1.5%. These conditions provided recoveries ≥ 92.8% and CV ≤ 5.8%. The experiments confirmed the predicted results, indicating that the optimized conditions and models used were effective in the determination of amines from milk. Samples of raw milk and milk beverages were observed to be poor sources of spermine and spermidine, whereas human milk contained both amines. High performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry for sensitive determination of bioactive amines in donkey milk In the present study we report on the optimization and validation of a sensitive high performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS) method for the determination of 8 bioactive amines (histamine, tyramine, tryptamine, 2-phenylethylamine, cadaverine, putrescine, spermidine and spermine) in donkey milk samples. The method involves donkey milk pre-treatment to remove proteins and pre-column dansylation of the amines. HPLC in reversed phase mode has been used for bioactive amines separation and the operating condition of the APCI-MS system proved to be powerful and very efficient for peak assignment. The separation was accomplished in a short time with an excellent resolution for all the amine peaks. Quantification was carried out by monitoring the characteristic [M+H](+) ion of each amine derivative. The method sensitivity, linearity and repeatability were assayed with satisfactory results. The detection limits of the analysed amines ranged from 0.5 microg L(-1) to 15 microg L(-1); the highest LOD was for spermine. Also remarkably good recovery values were obtained; at the lowest spiking level (1 microg L(-1)) the percent mean recoveries ranged from 77.7 to 109.7. Furthermore, as the investigations relate to a complex matrix as donkey milk, suitable studies on matrix effect were performed. Finally, the developed and validated method was applied to analyse 13 donkey milk samples. Among the identified bioactive amines, putrescine, spermine and spermidine proved to be the main amines in donkey milk. Their concentration levels in the present study were lower than the values determined in mature human, cow and sow milk. Polyamines in the Gastro-intestinal Tract of Goat Kids and in the Regenerating Ruminai Epithelium of Sheep The aim of the present study was to investigate whether temporal changes in polyamine concentration and synthesis could be found in the luminal content and wall tissue of the rumen and abomasum, two organs which have entirely different growth patterns during the first month of life. In the abomasal mucosa there was a marked gradual decrease in the ornithine decarboxylase (ODC) activity during the first month of life, while the ODC activity in the ruminal mucosa was low during the whole experimental period. However, injury of the rumen wall was followed by increased ODC activity. The ODC activity in duodenal mucosa was about 10 times higher than in the ileal mucosa and the ruminal epithelium. In ruminal liquid a clear peak in ODC activity was observed during the period 51-70 days after birth. The polyamine concentration did not parallel the ODC activity, in either the ruminal epithelium or the ruminal liquid. Of the polyamines, the spermine concentration was always highest, and with the exception of duodenal mucosa, the putrescine concentration was lowest. In liver a clear decrease in spermidine concentration from day 1 to about day 60 after birth was observed. Otherwise no marked temporal changes in tissue polyamine concentrations were observed. Two and a half hours after oral administration of 14C-labelled spermine, nearly all of the radioactivity was found in the lumen of the gastrointestinal tract. On the other hand, 1 h after intravenous injection of polyamines the walls of the gastrointestinal tract were strongly labelled. In conclusion, the polyamines needed for ruminal epithelial development seem to come from sources other than the ruminal epithelium itself or the ruminal lumen. View Inhibition of ornithine decarboxylase by α-difluoromethylornithine induces apoptosis of HC11 mouse mammary epithelial cells Feb 2000 T Płoszaj T Motyl W Zimowska Lech Zwierzchowski The effect of a-difluoromethylornithine (DFMO) on the apoptosis of HC11 mouse mammary epithelial cells was investigated. The involvement of reactive oxygen species (ROS) and Bcl-2 protein in the mechanism of apoptosis induced by ornithine decarboxylase (ODC) inhibition was also assessed. DFMO (0.1, 1 and 5mM) induced apoptosis of HC11 cells in dose- and time-dependent manner. Apoptosis manifests itself with morphological features like: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis (putrosis). The decrease in the nuclear DNA contents appearing as the hypodiploidal peak sub-G1 in the DNA histogram was not dependent on the presence of prolactin (5 microg/ml) in DFMO-treated cultures. Apoptosis induced by ODC inhibition was associated with a rapid increase in ROS concentration in HC11 cells observed within 1 h after DFMO treatment. The down-regulation of Bcl-2 as a decrease in cell number expressing bcl-2 and a lowered Bcl-2 protein content in cells expressing this protooncogene was also noted. The administration of putrescine (50 microM) lowered the number of early-apoptotic, late-apoptotic and necrotic cells. Moreover, it increased the number of cells expressing bcl-2. In conclusion, the disturbance of cellular polyamine homeostasis by inhibition of their synthesis enhances mammary epithelial cell susceptibility to apoptosis. It may occur in the mammary gland at the end of lactation, when the depletion of circulating lactogenic hormones and activation of intra-mammary apoptogenic factors expression take place. Analysis of polyamines as markers of (patho)physiological conditions Jan 2003 J CHROMATOGR B Diana Teti Maria Visalli Harold McNair The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators. Jun 1995 BRIT J NUTR Susan Bardocz Tracey J. Duguid David S. Brown Ann Ralph The polyamines putrescine, spermidine and spermine are essential for cell renewal and, therefore, are needed to keep the body healthy. It was previously believed that polyamines are synthesized by every cell in the body when required. However, in the present paper evidence is provided to show that, as in the case of the essential amino acids, the diet can supply sufficient amounts of polyamines to support cell renewal and growth. Systematic analysis of different foods was carried out and from the data obtained, the average daily polyamine consumption of British adults was calculated to be in the range 350–500 μmol/person per d. The major sources of putrescine were fruit, cheese and non-green vegetables. All foods contributed similar amounts of spermidine to the diet, although levels were generally higher in green vegetables. Meat was the richest source of spermine. However, only a part of the polyamines supplied by the diet is available for use by the body. Based on experiments with rats it was established that polyamines were readily taken up from the gut lumen, probably by passive diffusion, and were partly metabolized during the process of absorption. More than 80% of the putrescine was converted to other polyamines and non-polyamine metabolites, mostly to amino acids. The enzyme responsible for controlling the bioavailability of putrescine was diamine oxidase (EC 1.4.3.6). For spermidine and spermine, however, about 70–80% of the intragastrically intubated dose remained in the original form. Considering the limitations on bioavailability (metabolism and conversion), the amounts of polyamines supplied by the average daily diet in Britain should satisfy metabolic requirements. Effects of Pinealectomy on the Circadian Rhythms of the Activities of Polyamine Biosynthetic Decarboxylases and Tyrosine Aminotransferase in Different Organs of the Rat Mar 1979 Giuseppe Scalabrino Maria Elena Ferioli R Nebuloni Franco Fraschini The circadian rhythms of the activities of some enzymes were compared in liver, kidneys, and thymus of sham-pinealectomized and pinealectomized rats. Ornithine decarboxylase and tyrosine aminotransferase were chosen because of the well known fluctuations of their activities during the day. The diurnal behavior of S-adenosyl-L-methionine decarboxylase activity was first ascertained to display circadian rhythm in all of the above-mentioned organs of sham-pinealectomized rats and then was compared with that of pinealectomized rats. Before the operation and until killed, all of the animals were kept under rigidly standardized conditions. They were killed at 6-h intervals during the day. Chronobiologically in pinealectomized animals, the acrophase of the circadian rhythm of ornithine decarboxylase activity was markedly shifted in liver and, to a lesser degree, in kidneys. For the same enzyme, pinealectomy modified the amplitude in thymus and, even more so in kidneys. As for the circadian rhythm of S-adenosyl-L-methionine decarboxylase activity, pinealectomy induced an increase of the amplitude only in kidneys. No chronobiological parameter of the circadian rhythm of tyrosine aminotransferase activity was changed by pinealectomy in any of the chosen organs. The results of chronobiological analyses are supported by and in agreement with the results of analyses of variance of the effects of pinealectomy on the circadian rhythms tested. Two conclusions seem to be justified: 1) the pineal gland plays a part in the rat time-keeping machinery regulating enzyme circadian rhythms; and 2) in the circadian organization of the rat, the pineal gland plays the role of a coupling device, rather than that of master oscillator. Jun 1976 K Kano T Oka Mouse mammary gland has been shown to possess a transport system for spermidine, spermine, and putrescine. The uptake system for sperimidine, as studied in detail on mammary explants in culture is a time-dependent, energy-requiring process which can be stimulated by insulin and prolactin. The stimulatory effect of insulin involves both enhancement of Vmax for spermidine influx and prevention of efflux of the polyamine, whereas prolactin, in the presence of insulin, elicits a greater increase in Vmax for spermidine. Studies are also reported on the effects of temperature, concentration, and various inhibitors on this system. The accumulated spermidine exists virtually in an unchanged form with little metabolic conversion to either spermine or putrescine or to its conjugated form. In contrast, spermine and putrescine, both of which are also taken up by mammary explants, undergo metabolic conversion to spermidine. Sequestered end products and enzyme regulation: The case of ornithine decarboxylase Jul 1992 Microbiol Rev R H Davis David Robert Morris Philip Coffino The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools. Sequestered end products and enzyme regulation: the case of ornithine decarboxylase David Robert Morris Philip Coffino The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools. The multiple regulatory roles of polyamines in the hormonal induction of mammary gland development Article Jan 1981 T. Oka J. Perry T. Takemoto H. Inoue Polyamines in the Gastrointestinal Tract Article Jan 1992 Véronique Catros-Quemener J P Moulinoux C Bergeron Nadja Seiler Polyamine profile in ovine and caprine colostrum and milk The objective of this study was to monitor the post-partum variation of polyamine content, in ovine and caprine milk, from indigenous Greek breeds. Twenty samples of ewe and 20 samples of goat colostrum and milk were collected at the 1st, 2nd, 3rd, 4th, 5th and 15th day post-partum. Putrescine, spermidine and spermine were measured as dansylated derivatives by high-performance liquid ... [Show full abstract] chromatography. Putrescine was the least concentrated of these substances in both milk types. Spermidine was the prevailing polyamine in caprine samples, reaching levels up to 4.41 μmol/l on the 3rd day post-partum. In ovine milk, the profile of the mean concentrations showed greater levels of spermine than spermidine, except for the 5th day post-partum. These data suggest that goat colostrum and ewe milk (15th day) could be considered as good natural sources for these bioactive growth factors, and may become useful raw materials for designing tailored dairy products for specific population groups.	https://www.researchgate.net/publication/13812249_Polyamines_in_Goat's_Colostrum_and_Milk
PRIME PubMed \| Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans PubMed journal article: Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. Download Prime PubMed App to iPhone, iPad, or Android Tags Type your tag names separated by a space and hit enter Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. Int J Biol Macromol . 2021 Jan 15; 167:423-433. IJ Abstract The present study was designed to explore the in vivo-antioxidant capacity and the probable mechanism of AAPs-H, prepared from Auricularia auricula polysaccharides with the optimal extraction conditions by Box-Behnken design and acid hydrolysis, using Caenorhabditis elegans as a model organism. The effects of AAPs-H on the locomotion behavior, life span, antioxidant-related enzymes activities, and antioxidants levels in C. elegans were studied. Furthermore, the potentials of AAPs-H in up-regulating the expression of antioxidant-related genes in C. elegans, such as skn-1, sod-3 and sir-2.1, were also discussed. AAPs-H demonstrated a highly significant protective effect against the damage caused by paraquat, could significantly increase U-Turn frequency of worms (p < 0.01), extend their lifespan, enhance antioxidant systems including GR by 63.96% (p < 0.05), GSH-Px by 71.16% (p < 0.01), SOD by 78.65% (p < 0.01) and CAT by 98.52% (p < 0.01), increase the level of GSH by 28.12% (p < 0.05), and decrease the level of MDA by 39.29% (p < 0.01). The qRT-PCR results showed that AAPs-H could up regulate mRNA expression levels of skn-1, sod-1, sod-2, sod-3 and sir-2.1 in wild-type C. elegans (>1.6 fold) when treated with the concentration of 0.4 mg/mL (p < 0.05 or p < 0.01). Our studies provide evidence that AAPs-H improves antioxidant defense system, and up-regulation of oxidative stress related genes for prevention of stress damage in C. elegans. Links Publisher Full Text (DOI) Authors +Show Affiliations Gu J College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Li Q College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Liu J College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Ye Z College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Feng T College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Wang G College of Modern Science and Technology, China Jiliang University, Hangzhou, Zhejiang 310018, China. Wang W College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Zhang Y College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China. Electronic address: yjzhang@vip.163.com. MeSH Animals Antioxidants Auricularia Biological Products Caenorhabditis elegans Chemical Fractionation Fungal Polysaccharides Gene Expression Regulation Hydrolysis Locomotion Oxidative Stress Paraquat Reactive Oxygen Species Superoxide Dismutase Temperature Ultrasonic Waves Pub Type(s) Journal Article Language eng PubMed ID 33249158 Citation Gu, Jingyi, et al. "Ultrasonic-assisted Extraction of Polysaccharides From Auricularia Auricula and Effects of Its Acid Hydrolysate On the Biological Function of Caenorhabditis Elegans." International Journal of Biological Macromolecules, vol. 167, 2021, pp. 423-433. Gu J, Li Q, Liu J, et al. Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. Int J Biol Macromol . 2021;167:423-433. Gu, J., Li, Q., Liu, J., Ye, Z., Feng, T., Wang, G., Wang, W., & Zhang, Y. (2021). Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. International Journal of Biological Macromolecules , 167 , 423-433. https://doi.org/10.1016/j.ijbiomac.2020.11.160 Gu J, et al. Ultrasonic-assisted Extraction of Polysaccharides From Auricularia Auricula and Effects of Its Acid Hydrolysate On the Biological Function of Caenorhabditis Elegans. Int J Biol Macromol. 2021 Jan 15;167:423-433. PubMed PMID: 33249158. * Article titles in AMA citation format should be in sentence-case Copy Download MLA AMA APA VANCOUVER TY - JOUR T1 - Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. AU - Gu,Jingyi, AU - Li,Qiaowei, AU - Liu,Jing, AU - Ye,Zhongdu, AU - Feng,Tao, AU - Wang,Ge, AU - Wang,Weimin, AU - Zhang,Yongjun, Y1 - 2020/11/27/ PY - 2020/09/25/received PY - 2020/11/16/revised PY - 2020/11/23/accepted PY - 2020/11/30/pubmed PY - 2021/4/22/medline PY - 2020/11/29/entrez KW - Antioxidant systems KW - Antioxidant-related genes KW - Auricularia auricula polysaccharides KW - C. elegans model KW - Stress-resistance SP - 423 EP - 433 JF - International journal of biological macromolecules JO - Int J Biol Macromol VL - 167 N2 - The present study was designed to explore the in vivo-antioxidant capacity and the probable mechanism of AAPs-H, prepared from Auricularia auricula polysaccharides with the optimal extraction conditions by Box-Behnken design and acid hydrolysis, using Caenorhabditis elegans as a model organism. The effects of AAPs-H on the locomotion behavior, life span, antioxidant-related enzymes activities, and antioxidants levels in C. elegans were studied. Furthermore, the potentials of AAPs-H in up-regulating the expression of antioxidant-related genes in C. elegans, such as skn-1, sod-3 and sir-2.1, were also discussed. AAPs-H demonstrated a highly significant protective effect against the damage caused by paraquat, could significantly increase U-Turn frequency of worms (p < 0.01), extend their lifespan, enhance antioxidant systems including GR by 63.96% (p < 0.05), GSH-Px by 71.16% (p < 0.01), SOD by 78.65% (p < 0.01) and CAT by 98.52% (p < 0.01), increase the level of GSH by 28.12% (p < 0.05), and decrease the level of MDA by 39.29% (p < 0.01). The qRT-PCR results showed that AAPs-H could up regulate mRNA expression levels of skn-1, sod-1, sod-2, sod-3 and sir-2.1 in wild-type C. elegans (>1.6 fold) when treated with the concentration of 0.4 mg/mL (p < 0.05 or p < 0.01). Our studies provide evidence that AAPs-H improves antioxidant defense system, and up-regulation of oxidative stress related genes for prevention of stress damage in C. elegans. SN - 1879-0003 UR - https://www.unboundmedicine.com/medline/citation/33249158/Ultrasonic_assisted_extraction_of_polysaccharides_from_Auricularia_auricula_and_effects_of_its_acid_hydrolysate_on_the_biological_function_of_Caenorhabditis_elegans_ DB - PRIME DP - Unbound Medicine ER - Grapherence [↓11] Links Publisher Full Text (DOI) Authors Gu J Li Q Liu J Ye Z Feng T Wang G Wang W Zhang Y MESH Animals Antioxidants Auricularia Biological Products Caenorhabditis elegans Chemical Fractionation Fungal Polysaccharides Gene Expression Regulation Hydrolysis Locomotion Oxidative Stress Paraquat Reactive Oxygen Species Superoxide Dismutase Temperature Ultrasonic Waves Coronavirus Guidelines Latest evidence on COVID-19 from PubMed, WHO, CDC. Visit free Relief Central . Try the Free App: Prime PubMed is provided free to individuals by: Unbound Medicine . Grapherence [↓11] Related Citations Evaluation of the antioxidant effects of acid hydrolysates from Auricularia auricular polysaccharides using a Caenorhabditis elegans model. Optimization of the polysaccharide hydrolysate from Auricularia auricula with antioxidant activity by response surface methodology. The lipid lowering and antioxidative stress potential of polysaccharide from Auricularia auricula prepared by enzymatic method. Comparison on chemical features and antioxidant activity of polysaccharides from Auricularia auricula by three different enzymes. Comparison of the chemical composition and antioxidant stress ability of polysaccharides from Auricularia auricula under different drying methods. Antioxidant activity in vitro and in vivo of the polysaccharides from different varieties of Auricularia auricula. Effect of Enzyme-Assisted Extraction on the Chemical Properties and Antioxidant Activities of Polysaccharides Obtained from the Wood Ear Mushroom, Auricularia auricula (Agaricomycetes). Polysaccharides from Auricularia auricula: Preparation, structural features and biological activities. Effects of the Extraction Solvents on Dissolution Rate and Antioxidant Capacity of Auricularia auricula (Agaricomycetes) Polysaccharides In Vitro and In Vivo. Hypoglycemic Effect of the Degraded Polysaccharides from the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Agaricomycetes). More Home Contact Us Help Privacy / Disclaimer Terms of Service Sign In	https://www.unboundmedicine.com/medline/citation/33249158/Ultrasonic_assisted_extraction_of_polysaccharides_from_Auricularia_auricula_and_effects_of_its_acid_hydrolysate_on_the_biological_function_of_Caenorhabditis_elegans_
Multi-layered enzyme coating on highly conductive magnetic biochar nanoparticles for bisphenol A sensing in water \| Request PDF Request PDF \| Multi-layered enzyme coating on highly conductive magnetic biochar nanoparticles for bisphenol A sensing in water \| This work introduced stable immobilization of tyrosinase (TYR) enzymes onto the highly conductive magnetic biochar nanoparticles with carboxyl... \| Find, read and cite all the research you need on ResearchGate October 2019 Chemical Engineering Journal 384:123276 DOI: 10.1016/j.cej.2019.123276 Authors: Lingzhi He Huazhong University of Science and Technology Yuesuo Yang Jilin University Jungbae Kim Korea University Lan Yao Lan Yao This person is not on ResearchGate, or hasn't claimed this research yet. Show all 7 authors Hide Request full-text Download citation Copy link Link copied Request full-text Download citation Copy link Link copied To read the full-text of this research, you can request a copy directly from the authors. Citations (48) References (48) Abstract This work introduced stable immobilization of tyrosinase (TYR) enzymes onto the highly conductive magnetic biochar nanoparticles with carboxyl functionality to construct sensitive and reusable electrochemical biosensor for bisphenol A (BPA) detection. The newly synthesized biochar nanoparticles (BCNPs) from biomass had outstanding electrochemical activity, after functionalization with carboxylic functional groups and magnetic nanoparticles (Mag-BCNPs-COOH), it was used for multi-layered TYR immobilization with covalent attachment, precipitation and cross-linking method (ML-TYR/Mag-BCNPs-COOH). The BPA sensing system was simply constructed by directly attaching the biocatalyst of ML-TYR/Mag-BCNPs-COOH onto a glassy carbon electrode with magnet force. Owning to great properties of the ML-TYR/Mag-BCNPs-COOH, such as high stability, biocatalytic activity and conductivity, the BPA sensing signal was significantly enhanced. And the lowest detection limit of 2.78 nM with linear ranges from 0.01 to 1.01 μM was achieved. The modified electrode could be reused at least eight cycles without reducing the sensing signal, and could be used for the real environmental water detection. Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations Join for free No full-text available To read the full-text of this research, you can request a copy directly from the authors. Request full-text PDF Citations (48) References (48) ... Additionally, biochar nanoparticles (BCNPs) can also exhibit excellent electrocatalytic properties as well as a large surface area and have been well-developed in electrochemical biosensing. 177, 178 Quantum dots are a type of nanomaterial with a size less than 100 nanometers. Graphene quantum dots (GQDs) 156 and palladium quantum dots (PdQDs) 163 have been used to modify the electrode due to their good water solubility and biocompatibility. ... ... 182 SAMs are sometimes introduced onto metal electrodes, where the functional terminals are able to couple with receptors, for instance, cysteamine grown on a gold surface to support MIP, 169 mercaptoacetic acid fixed on a gold surface for antibody immobilization, 135 and mercaptoundecanoic acid (11-MUA) grafted at a silver surface for the surface anchoring of an antibody. 137 Serving as a specific organic linker for carboxylic groups, ethyl (dimethylamino propyl) carbodiimide (EDC) 140 or ethyl (dimethylamino propyl) carbodiimide/Nhydroxysuccinimide (EDC/NHS) 135,137,144, 178 is often included in an experiment to achieve activation and protection from carboxylic radicals. However, based on the conclusion of Zhang et al., 114 Liang et al. 134 utilized graphene oxide nanoribbons/multi-walled carbon nanotubes (GONRs/ MWCNTs) hybrids to implant antigens without using linkage chemicals. ... ... The reduction of o-quinone forms an enzymatic reaction loop to regenerate catechol, yielding reductive currents that indicate the concentrations of BPA. 125,[177] [178] [179] Lac is different from Tyr as it belongs to a multi-copper-containing oxidase family. Lac can be obtained from various species of fungi, plants, insects, and bacteria. ... Endocrine Disrupting Chemicals in Water and Recent Advances on Their Detection Using Electrochemical Biosensors Article Full-text available Dec 2022 Lue Wang Haoyun Wang Chedly Tizaoui Wei Zhang The ever increasing anthropogenic activities have been producing an undesired group of substances called endocrine disrupting chemicals (EDCs), which can pose a serious threat to the health of human beings and wildlife once they are released into natural water environments. Herein, recent advances on the use of electrochemical biosensors for the determination of EDCs especially in water are thoroughly summarized. Initially, different categories of EDCs with important guidelines are briefly introduced, followed by a simple discussion of non-electrochemical detection methods. Electrochemical techniques including theoretical background and reported studies are then evaluated as the highlight of the review. Finally, current issues, technical bottlenecks, and prospects of this field are critically discussed. This review is composed of following subsections: (i) systematical classification of common EDCs with their toxicities towards human beings and animals, (ii) water contamination events, safety guidelines, and legislations, (iii) non-electrochemical methods for detection of EDCs, (iv) electrochemical monitoring systems, (v) receptors for biorecognition, (vi) detection of EDCs using electrochemical biosensors, and (vii) recent progress, issues, and further development. This review, with a strong interdisciplinary nature, across nanotechnology, biology, material science, and electrochemistry, can offer comprehensive academic assistance for future studies on the detection of EDCs using electrochemical biosensors. View Show abstract ... In this work (the general scheme is reported in Figure 4), a stable immobilization of the tyrosinase (Tyr) enzyme using the carboxyl functionality of the highly conductive magnetic biochar nanoparticles was proposed. This biosensor, based on GCEs, presented very promising analytical performances with a detection limit (LOD) of 2.78 nM with linear ranges from 0.01 to 1.01 µM [115] . and the electrocatalytic capacity of biochar to develop a voltammetric sensor for the determination of lead (II) and cadmium (II). ... ... Another remarkable application is that of L. He and coworkers (2020). They developed a sensitive and reusable electrochemical biosensor for bisphenol A (BPA) based on newly synthesized biochar nanoparticles (BCNPs) [115] . In this work (the general scheme is reported in Figure 4), a stable immobilization of the tyrosinase (Tyr) enzyme using the carboxyl functionality of the highly conductive magnetic biochar nanoparticles was proposed. ... ... In this work (the general scheme is reported in Figure 4), a stable immobilization of the tyrosinase (Tyr) enzyme using the carboxyl functionality of the highly conductive magnetic biochar nanoparticles was proposed. This biosensor, based on GCEs, presented very promising analytical performances with a detection limit (LOD) of 2.78 nM with linear ranges from 0.01 to 1.01 μM [115] . Moreover, this is not the only biochar-based biosensor for the detection of bisphenol A. Indeed, Y. Liu and coworkers (2019) presented an electrochemical tyrosinase enzyme (Tyr)-based biosensor using a highly conductive sugarcane-derived biochar nanoparticle (BCNP) for the detection of BPA [110]. ... Biochar: A Sustainable Alternative in the Development of Electrochemical Printed Platforms Article Full-text available Aug 2022 Rocco Cancelliere Miriam Cianciaruso Katya Carbone Laura Micheli Biochar is a pyrolytic material with several environmental benefits such as reducing greenhouse gas emissions, sequestering atmospheric carbon and contrasting global warming. However, nowadays, it has moved to the forefront for its conductivity and electron transfer properties, finding applications in the fabrication of electrochemical platforms. In this field, researchers have focused on low-cost biomass capable of replacing more popular and expensive carbonaceous nanomaterials (i.e., graphene, nanotubes and quantum dots) in the realization of sensitive cost-effectiveness and eco-friendly electrochemical tools. This review discusses recent developments of biochar-modified screen-printed electrodes (SPEs). Special attention has been paid to biochar’s manufacturing processes, electron-donating capabilities and sensing applications. Examples of representative works are introduced to explain the distinct roles of biochar in several electro-bioanalytical strategies. View Show abstract ... As shown in Figure 4, He et al., prepared BCNPs from bagasse waste showing great electrochemical activity, and after functionalization with carboxylic functional groups and magnetic nanoparticles (Mag-BCNPs-COOH), the preparation was used for multi-layered TYR immobilization with a covalent attachment, precipitation, and cross-linking method (ML-TYR/Mag-BCNPs-COOH) [43] . Owing to the great properties of the ML-TYR/Mag-BCNPs-COOH, the prepared electrochemical biosensor was used for the detection of bisphenol A (BPA), and an LOD of 2.78 nM with linear ranges from 0.01 to 1.01 μM was obtained. ... ... As shown in Figure 4, He et al., prepared BCNPs from bagasse waste showing great electrochemical activity, and after functionalization with carboxylic functional groups and magnetic nanoparticles (Mag-BCNPs-COOH), the preparation was used for multi-layered TYR immobilization with a covalent attachment, precipitation, and cross-linking method (ML-TYR/Mag-BCNPs-COOH) [43] . Owing to the great properties of the ML-TYR/Mag-BCNPs-COOH, the prepared electrochemical biosensor was used for the detection of bisphenol A (BPA), and an LOD of 2.78 nM with linear ranges from 0.01 to 1.01 µM was obtained. ... ... The selectivity of the biosensor toward 3 µM BPA in the presence of various ionic interferents (with 100-fold concentration higher than BPA), such as Cl − , Ca 2+ , SO 4 2− , and Mg 2+ , was assessed and the detection of BPA was not affected. In another recent work reported by He et al., they immobilized tyrosinase enzymes onto highly conductive magnetic biochar nanoparticles (BCNPs) with carboxyl functionality (Mag-BCNPs-COOH) by covalent, cross-linking, and precipitation methods to fabricate a sensitive and reusable electrochemical biosensor for the detection of BPA [43] . It was found that the composite has good electrochemical activity, and the multilayer fixed composite (ML-TYR/Mag-BCNPs-COOH) had a much higher current response to BPA than did a single layer (SL-TYR/Mag-BCNPs-COOH). ... Recent Advances of Biochar-Based Electrochemical Sensors and Biosensors Article Full-text available May 2022 Yunxiao Li Rui Xu Huabin Wang Yong Zhang In the context of accelerating the global realization of carbon peaking and carbon neutralization, biochar produced from biomass feedstock via a pyrolysis process has been more and more focused on by people from various fields. Biochar is a carbon-rich material with good properties that could be used as a carrier, a catalyst, and an absorbent. Such properties have made biochar a good candidate as a base material in the fabrication of electrochemical sensors or biosensors, like carbon nanotube and graphene. However, the study of the applications of biochar in electrochemical sensing technology is just beginning; there are still many challenges to be conquered. In order to better carry out this research, we reviewed almost all of the recent papers published in the past 5 years on biochar-based electrochemical sensors and biosensors. This review is different from the previously published review papers, in which the types of biomass feedstock, the preparation methods, and the characteristics of biochar were mainly discussed. First, the role of biochar in the fabrication of electrochemical sensors and biosensors is summarized. Then, the analytes determined by means of biochar-based electrochemical sensors and biosensors are discussed. Finally, the perspectives and challenges in applying biochar in electrochemical sensors and biosensors are provided. View Show abstract ... A membrane separation is required for applications with special requirements on particle sizes. For instance, when used as electrodes for chemical detection, the size of nano-biochar particles is preferable to be < 220 nm [30, 40] . In some cases, membrane filtration is suggested to be a post step after sedimentation, by which most large particles can be removed, to ensure the filtration efficiency and improve the reusability of the membrane. ... ... The conductivity of electrodes was enhanced with transfer resistance reduced from 495 Ω for the pristine electrode to 325 Ω for electrodes loaded with nano-biochar prepared at 800 • C. He et al. [40] loaded tyrosinase (TYR), an enzyme from a mushroom with high sensitivity to BPA, on magnetic nano-biochar, and used it for BPA detection. The TYR/nano-biochar electrochemical sensor achieved the lowest detection limit of 2.78 nM with a linear range between 0.01 and 1.01 μM, and the sensibility maintained stable after 8 cycles without reducing the signal. ... ... Particle size distribution (A)[29][30][31][32][33][34][35][36][37][38] and average particle size (B)[30,[39] [40] [41][42][43][44][45][46] of reported nano-biochar in references. FB: fruit tree branches, CB: corn straw, PB: peanut straw, SS: sewage sludge, PM: pig manure, DM: dairy manure, RH: rice husk, PS: peanut shell, BG: barley grass, WS: wheat straw, PW: pine wood, WC: wood chip, BS: bagasse, CNS: coconut shell, WD: wood sawdust, TB: oak tree branches, CS: corn straw, WD: woody, CM: cattle manure, SB: soybean straw, BS− COOH: functionalized BS, NC: corn straws, NW: wheat, NR: rice, RS: rice straw. ... Preparation of nano-biochar from conventional biorefineries for high-value applications Article Apr 2022 RENEW SUST ENERG REV Bing Song Cao Xuewen Wenran Gao Richen Lin Nano-biochar presents superior physicochemical properties over bulk biochar such as highly stable and unique nanostructure, high catalytic activity and large specific surface area. A comprehensive understanding of the current nano-biochar research is required while progressing to real-world implementations, including preparation, characteristic properties and targeted applications. This paper provides a state-of-the-art review on nano-biochar research for (1) definition and production methodologies, (2) compositions and properties, (3) high-value applications and performances, (4) environmental concerns, and (5) implications to conventional biorefineries and perspectives. Following a sequence of biochar preparation, size reduction and screening, nano-biochar can be obtained for high-value applications such as a high-performance adsorbent, a carrier of biocatalysts, electrodes in electrochemistry and additives. Compared with bulk biochar, nano-biochar performs better in general uses such as wastewater treatment and soil amendment and promises high-value applications in emerging areas such as electrochemistry and biocatalysis. This review highlights that the preparation of nano-biochar from conventional bioenergy and biorefinery technologies such as thermochemical liquefaction and gasification is promising to generate adding values, contributing to enhanced techno-economic feasibility. Integrating nano-biochar preparation and application with conventional biofuel production is thus highly recommended as a future research endeavour. View Show abstract ... Acidic activation (by e.g. HNO 3 or H 2 SO 4 ) promotes the production of porous structures but it also leads to the formation of oxygen-containing functional groups at the biochar surface [30, 31] . Finally, metal salts and oxides (e.g. ... ... Besides metal/metal oxides decoration, biochar can be also easily modified with enzymes to fabricate selective biosensors [31, 40,48,49]. The modification process is extremely facile and fast: biochar is soaked in the enzyme solution, incubated for some hours and finally washed with water [31,48]. ... ... Besides metal/metal oxides decoration, biochar can be also easily modified with enzymes to fabricate selective biosensors [31,40,48,49]. The modification process is extremely facile and fast: biochar is soaked in the enzyme solution, incubated for some hours and finally washed with water [31, 48]. In this case, the enhancement in terms of sensitivity and selectivity provided by the decorating agent is ascribed to a biocatalytic effect: the bioreceptor (enzyme) recognizes the analyte and catalyzes a reaction leading to consumption of the analyte. ... Biochar as an alternative sustainable platform for sensing applications: A review Article Full-text available Sep 2020 Davide Spanu Gilberto Binda Carlo Dossi Damiano Monticelli Biomasses are emerging as alternatives to traditional synthetic carbon nanostructures to produce greener, carbon based, sensing platforms. More recently, biochar, a porous carbonaceous material produced by direct (i.e. solvent free) pyrolysis of biomasses, was also investigated, leading to a further decrease in the fabrication environmental footprint. Moreover, valuable byproducts (biogas and bio-oil) are also produced, in line with the atom economy principle of green chemistry. Here we review more than 100 papers published in the last five years describing biochar-based sensing platforms. The fabrication processes, tailoring and analytical applications of biochar-based sensors are presented and discussed aiming at shedding light on biochar potentialities in replacing less sustainable traditional materials. Electrochemical platforms were mainly developed (70% of the published papers), but also optical and other sensing mechanisms (electrogenerated luminescence and resistivity) were exploited. Analytical performances for biochar-based sensors are often reported as comparable to those of the best carbon-based sensing platforms, mainly thanks to the very high specific surface area and the presence of reactive surface functional groups (self-doping). Biochar decoration with metal/metal oxide nanoparticles and enzymes is also employed to further improve its sensing properties. Notwithstanding biochar applications were only recently introduced and mechanisms behind their selectivity and sensitivity are still under investigation, their analytical performances and greenness foster biochar employment as sensing platform. View Show abstract ... In this work, the multi-layered enzymatic immobilization was carried out through covalent bonding, cross-linking, and precipitation. The immobilization of the enzyme led to better stability, biocatalytic activity, and conductivity with significant enhancement in the sensing signal (He et al., 2020) . ... ... In another study, laccase was immobilized on wheat straw biochar using adsorption and cross-linking towards the removal of 2,4-DCP from the soil which led to better enzymatic stability (Wang et al., 2019). Also, TYR was immobilized on biochar nanoparticles using covalent bonding, precipitation, and cross-linking for the development of a BPA biosensor (He et al., 2020) . GLU crosslinking was used for the immobilization of horseradish peroxidase (HRP) on chitosan-halloysite hybrid nanotubes (HNTs-CTS) for phenol removal from wastewater. ... Enzyme immobilization on biomass-derived carbon materials as a sustainable approach towards environmental applications Article Jul 2022 CHEMOSPHERE Geethanjali Bijoy Rijo Rajeev Libina Benny Anitha Varghese Enzymes with their environment-friendly nature and versatility have become highly important ‘green tools’ with a wide range of applications. Enzyme immobilization has further increased the utility and efficiency of these enzymes by improving their stability, reusability, and recyclability. Biomass-derived matrices when used for enzyme immobilization offer a sustainable solution to environmental pollution and fuel depletion at low costs. Biochar and other biomass-derived carbon materials obtained are suitable for the immobilization of enzymes through different immobilization strategies. Environmental pollution has become an utmost topic of research interest due to an ever-increasing trend being observed in anthropogenic activities. This has widely contributed to the release of various toxic effluents into the environment in their native or metabolized forms. Therefore, more focus is being directed toward the utilization of immobilized enzymes in the bioremediation of water and soil, biofuel production, and other environmental applications. In this review, up-to-date literature concerning the immobilization and potential uses of enzymes immobilized on biomass-derived carbon materials has been presented. View Show abstract ... In addition, Martins et al. [37], developed a biochar-based electrochemical immunosensor for the selective detection of Hantavirus Araucaria nucleoprotein, known as a common virus of wild rodents that can cause hemorrhagic fever in humans after contamination. Activated biochar has also been reported in electroanalytical sensors towards bisphenol A [38, 39] and ammonia detection [40]. ... ... developed a biochar-based electrochemical immunosensor for the selective detection of Hantavirus Araucaria nucleoprotein, known as a common virus of wild rodents that can cause hemorrhagic fever in humans after contamination. Activated biochar has also been reported in electroanalytical sensors towards bisphenol A [38, 39] and ammonia detection [40]. ... Acetylcholinesterase Biosensor Based on Functionalized Renewable Carbon Platform for Detection of Carbaryl in Food Article Full-text available Jul 2022 Erik W. Nunes Jesus Rascon Martin Kássio Leme da Silva Ivana Cesarino Enzymatic electrochemical biosensors play an important role in the agri-food sector due to the need to develop sustainable, low-cost, and easy-to-use analytical devices. Such biosensors can be used to monitor pathogens, endocrine disruptors, and pesticides, such as carbaryl, widely used in many crops. The use of renewable carbon (RC) sources, provided from biomass pyrolysis has been often applied in the fabrication of such sensors. This material is a great candidate for biosensor fabrication due to the presence of surface functional groups, porosity, and moderate surface area. This work describes the functionalization of RC material through an acid treatment with a sulfonitric solution HNO3/H2SO4 (1:3) and the resulting material was characterized by scanning electron microscopy. The obtained RC functionalized (RCF) and the acetylcholinesterase enzyme (AChE) were applied in the construction of the electrochemical biosensor on glassy carbon (GC) electrode and used to detect carbaryl in apple samples. The GC/RCF/AChE biosensor was able to detect the carbaryl pesticide from 5.0 to 30.0 nmol L−1, displaying a LOD of 4.5 nmol L−1. The detection of carbaryl in apple samples presented recoveries between 102.5 to 118.6% through the standard addition method. The proposed biosensor is a promising renewable tool for food safety. View Show abstract ... Biochar has also been used in fabrication of biosensor to detect trace level of organic contaminants via enzyme reaction. He et al. (He et al. (2020) ) synthesized a biosensor by enzyme coating on highly conductive magnetic biochar that was able to detect bisphenol A as low as 2.78 nM. In another study, Wang et al. (Wang et al. (2020)) prepared gold nanoparticles decorated biochar and used it as a biosensor for detecting hydroquinone and catechol in water. ... ... Biosensing material for detecting Bisphenol A Detection limit of 2.78 nM with linear ranges from 0.01 to 1.01 μM (He et al., 2020) Gold decorated ginger seedling biochar/-HAuCl 4 treatment of biochar Biosensing material for detecting hydroquinone and catechol ... A Review of Recent Advancements in Utilization of Biomass and Industrial Wastes into Engineered Biochar Article Jun 2020 J HAZARD MATER Gihoon Kwon Amit Bhatnagar Hailong Wang Hocheol Song For past few years, biochar has gained a great deal of attention for its versatile utility in agricultural and environmental applications. The diverse functionality and environmental-friendly nature of biochar have motivated many researchers to delve into biochar researches and spurred rapid expansion of literature in recent years. Biochar can be produced from virtually all the biomass, but the properties of biochar are highly dependent upon the types of feedstock biomass and preparation methods. The overall performances of as-prepared biochar in treating soil and water contaminants is generally inferior to activated carbon due to its lower surface area and limited functionalities. This limitation has led to many follow-up studies that focused on improving material characteristics by imparting desired functionality. Such efforts have greatly advanced knowledge to produce better-performing engineered biochar with enhanced capability and versatility. To this end, this review was prepared to compile recent advancements in fabrication and application of engineered biochar, especially with respect to the influences of biomass feedstock on the properties of biochar and the utilization of industrial wastes in fabrication of engineered biochar View Show abstract ... This tool exploited the ability of acid-activated biochar to interact with the aromatic molecule of MP by (π-π interaction) [50]. Bisphenol A in the same matrix in comparison of 3.78 nM of the previous biosensor [55] . ... ... Scheme of Biochar nanoparticles modification for the detection of BPA, using glassy carbon electrode [55] . B) Modification procedure of screen-printed electrode with Biochar dispersion [56]. ... Sustainable materials for the design of forefront printed (bio)sensors applied in agrifood sector Article May 2020 TRAC-TREND ANAL CHEM Fabiana Arduini Laura Micheli Viviana Scognamiglio Danila Moscone The search for sustainability has now become a duty for all those entities, nations or people who intend to combat poverty in the world, safeguard the environment and bridge the gap of disparities that unfortunately still exist between underdeveloped countries and medium-high developed nations. The agrifood sector plays an important role in this vision, and the development of sustainable analytical devices at low cost and easy to use for everyone, suitable for being adopted by those countries whose technological development does not allow them to take up expensive and sophisticated laboratory techniques, may constitute a contribution towards achieving the objectives listed above. In this review we illustrate some examples of electrochemical devices realized utilizing sustainable materials, such as paper, as support for disposable and reagent-free (bio)sensors applied to the monitoring of food quality and safety. We also mention the first applications of new carbonaceous materials obtained from the recycling of agrifood waste products, which promise interesting features for electrochemical applications. View Show abstract ... However, following enzyme immobilization, the surface of LmGACs seemed smoother, probably due to enzyme coating on the biochar surface [51], as illustrated in Fig. S1. Enzyme immobilization in magnetic biochar nanoparticles has been linked to a smoothing of the surface and a coating-like shape [13] . ... Removal of emerging pollutants from water using enzyme-immobilized activated carbon from coconut shell Article Full-text available Mar 2023 Osamah J. Al-sareji Mónika Meiczinger Viola Somogyi Khalid Hashim View ... Compared with MBC, LC-MBC surface became smoother after laccase immobilization. This phenomenon could be attributed to the presence of a thick layered enzyme coating on the surface (He et al., 2020) . The EDS analysis (Fig. 2(c)(f)) revealed C, O, N and Fe were the dominate elements of MBC and LC-MBC. ... Magnetically separable laccase-biochar composite enable highly efficient adsorption-degradation of quinolone antibiotics: Immobilization, removal performance and mechanisms Article Full-text available Mar 2023 SCI TOTAL ENVIRON Mengyuan Zou Weijun Tian Meile Chu Dongpo Xu The tremendous potential of hybrid technologies for the elimination of quinolone antibiotics has recently attracted considerable attention. This current work prepared a magnetically modified biochar (MBC) immobilized laccase product named LC-MBC through response surface methodology (RSM), and LC-MBC showed an excellent capacity in the removal of norfloxacin (NOR), enrofloxacin (ENR) and moxifloxacin (MFX) from aqueous solution. The superior pH, thermal, storage and operational stability demonstrated by LC-MBC revealed its potential for sustainable application. The removal efficiencies of LC-MBC in the presence of 1 mM 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) for NOR, ENR and MFX were 93.7 %, 65.4 % and 77.0 % at pH 4 and 40 °C after 48 h reaction, respectively, which were 1.2, 1.3 and 1.3 times higher than those of MBC under the same conditions. The synergistic effect of adsorption by MBC and degradation by laccase dominated the removal of quinolone antibiotics by LC-MBC. Pore-filling, electrostatic, hydrophobic, π-π interactions, surface complexation and hydrogen bonding contributed in the adsorption process. The attacks on the quinolone core and piperazine moiety were involved in the degradation process. This study underscored the possibility of immobilization of laccase on biochar for enhanced remediation of quinolone antibiotics-contaminated wastewater. The proposed physical adsorption-biodegradation system (LC-MBC-ABTS) provided a novel perspective for the efficient and sustainable removal of antibiotics in actual wastewater through combined multi-methods. View Show abstract ... Chaithra et al. [37] were able to synthesize biomass-derived carbon nanospheres from the leaves of Mimosa pudica, which were then incorporated with Pd nanoparticles for the electrochemical sensing of morin. Furthermore, there were recent reports of using biochar derived from sugarcane stalks which were successfully incorporated into biosensors [38, 39]. Recent studies have also shown that sugarcane bagasse can be used as a sustainable bio-sorbent to remove methylene blue (MB) from wastewater polluted with the compound [40][41][42]. ... Methylene Blue-Modified Biochar from Sugarcane for the Simultaneous Electrochemical Detection of Four DNA Bases Article Full-text available Mar 2023 Qusai Hassan The abnormal levels of four DNA bases, namely guanine (G), adenine (A), thymine (T), and cytosine (C) are implicated in several cancers, metabolic diseases, and HIV/AIDS. Therefore, the accurate detection and concentration measurement of these four DNA bases is of significant interest. Furthermore, there has recently been a push towards developing chemical sensors which are more sustainable and cost-effective. Herein, we developed a graphite paste electrode which incorporated the biochar of sugarcane and methylene blue (GPE-SC-MB) in order to simultaneously detect these four DNA bases. The linear ranges obtained for the four DNA bases are 0.67–38.67 µM for G, 0.67–126.67 µM for A, and 6.67–1600 µM for T and C. The limit-of-detection (LOD) values obtained were 0.037 μM for G, 0.042 µM for A, 4.25 μM for T, and 5.33 µM for C. The electroactive surface area of the electrode as well as the diffusion coefficients for each analyte were determined. Lastly, the GPE-SC-MB was tested in real samples using human saliva with recovery values between 99.0 and 103.0%. Thus, biochar from sugarcane proved to be an effective electrode modifier material for the development of sensitive electrochemical sensors. View Show abstract ... Figure 3C shows the O 1 s spectra at 531 eV, 531.62 eV and 532.48 eV corresponding to metal oxygen (ZnO), C=O and C-O/OH. Figure 3D shows the Zn 2p spectrum at 1022 eV and 1045.1 eV corresponding to 2p 3/2 and 2p 1/2 orbitals, respectively [26, 27] . These data by XPS also illustrates the successful synthesis of the ZnO/BC nanohybrid. ... Green Synthesis of ZnO/BC Nanohybrid for Fast and Sensitive Detection of Bisphenol A in Water Article Full-text available Apr 2022 Jiafeng Hu Dongpeng Mao Penghu Duan Yunxian Piao A nanohybrid of zinc oxide and biochar (ZnO/BC) with high conductivity was green synthesized using a simple hydrothermal method, and utilized for the sensitive detection of bisphenol A (BPA) by coating the nanohybrid film on an electrode of glassy carbon. The ZnO/BC presented greatly improved electrocatalytic performance and electron transfer ability compared to the zinc oxide and biochar. The ZnO/BC film-coated electrode could detect the BPA in aqueous solution within 3 min while neglected interference from higher concentrations of regularly existing ions and similar concentrations of estradiol (E2), phenol, dichlorophenol (DCP), and ethinylestradiol (EE2). Under optimal conditions, the linear range of BPA detection was 5 × 10−7~1 × 10−4 mol/L, with a detection limit of 1 × 10−7 mol/L, and the detection sensitivity was 92 mA/M. In addition, the ZnO/BC electrode could detect BPA in a real water sample with good signal recovery. This electrode, with the advantages of an easy preparation, low cost, and fast response time, could be potentially applicable for environmental monitoring. View Show abstract ... piao group reported a bisphenol a (Bpa) sensor in water using highly conductive magnetic biochar. it claims the proposed sensing can be used up to 8 cycles during eight weeks with excellent repeatability performance [46] . The microfluidic chip is well known for disposable chip after the measurement; interestingly, a reusable 3-d printed microfluidic was proposed by hondred et al. it is integrated with the reliable electrode tape with the capability of several hundred bending cycles [47]. in addition, a flexible and reusable electrode sheet of isfeT sensor was proposed for ph sensing measurement. ... Environmentally Friendly and Biodegradable Components for Biosensors Article Oct 2022 Briliant A. Prabowo Agnes Purwidyantri The pandemic of coronavirus diseases 2019 (covid-19) attracts dramatic attention worldwide that biosensing technology plays an essential role in screening, tracing, and diagnostics of the diseases in society. The World health organization (Who) recommends the significant features of biosensor development, called assured: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and delivered to the end-user. consequently , the practical, low-cost, and environmentally friendly components of biosensors are urgently required in the future for such cases of massive tests in homecare and point of care. This article reviews the emerging trend and applications View Show abstract ... In the future, the addition of nanocomposites, and thermal resistive DNA nanomaterial combined with enzymes, can be beneficial for improving the efficiency of biosensors [72]. In addition, these nanocomposites will be used to immobilize distinct types of a diverse range of enzymes, towards the detection of multiple biomolecules via biosensors, with a high sensitivity [73] . ... Enzyme Immobilized Nanoparticles Towards Biosensor Fabrication Chapter Jun 2022 Jaison Jeevanandam Sharadwata Pan Michael K. Danquah In recent times, nanomaterials with semiconductor properties are introduced as a potential transducer in biosensors, which can be credited to their intrinsic, elevated surface-to-volume proportion, enhanced sensitivity, and improved surface properties. The surface properties of nanomaterials have made them a significant transducer matrix towards the immobilization of bioreceptors, which eventually enhances the identification threshold and the biosensor sensing capability. Several nanomaterials, such as polymer, metal oxide, metal and carbon-based, as well as nanocomposites, are used towards transducer manufacturing, eventually being incorporated in the biosensors. The current chapter lays an outline with respect to biosensors that are fabricated with nanomaterials as a transducer, where enzymes acting as a bioreceptor, are immobilized on their surface. In addition, the biosensing mechanisms of the enzyme immobilized nanomaterials, their efficiency, detection limit, and sensitivity, are also discussed. View Show abstract ... Upon reacting with tyrosinase, tyrosinase is enriched and well scattered on AArGO (i.e., Tyr/AArGO, Fig. 3(f)), which can be attributed to an enhanced grafting of tyrosinase due to higher content of -COOH groups on AArGO, as observed by XPS. It has been reported that more enzymes can be loaded on -COOH-functionalized biochar nanoparticles to enhance biocatalytic activity (He et al., 2020) . Nonetheless, in the later electrochemical performance section (Fig. 4(c)), we demonstrate that full coverage with tyrosinase hinders the conductive feature of AArGO. ... Environmentally benign and biocompatible sensing platform for electroanalytical determination of bisphenol A in the aquatic environment Article Sep 2022 Ting-Wei Lee Sing-Jen Jiang Bryan Gencianeo Alamani Chiaying Chen In this work, a facilely fabricated enzymatic biosensor composed of reduced graphene oxide (rGO), chitosan (Chi), and tyrosinase (Tyr) is developed for bisphenol A (BPA) determination. rGO with varying properties is obtained by different GO reduction routes. A trend correlating the sensing efficiency toward BPA with the population and distribution of surface oxygen functional groups on rGO is identified. Compared to other reducing routes, rGO fabricated using ascorbic acid, an environmentally benign reductant, has a lower amount of oxidation debris and higher contents of carboxyl groups. The increased content of carboxyl carbon in ascorbic acid-reduced graphene oxide (AArGO) contributes to promoting amide linkage (i.e., formation of more N-(C=O) groups) with chitosan, benefitting the assembly with tyrosinase to enhance BPA determination performance. The proposed Tyr/Chi/AArGO-modified electrodes serve to benefit rational designs of graphene-based nanocomposites for BPA determination and realize an environmentally benign, biocompatible, and facilely fabricated sensing platform for emerging contaminants in the aquatic environment. View Show abstract ... In the cross-linking method, multifunctional reagents like glutaraldehyde are used to immobilize the enzymes directly to the transducers [67,68]. For coupling with amino acids, these reagents have functional groups like -OH, -SH, -COOH, -NH 2 , etc. [69] . For example, Bilal et al. used ethylene glycol-bis [succinic acid N-hydroxysuccinimide, (EG-NHS)], which is comparable with glutaraldehyde as a linking agent for enzymes, and observed enhanced stability [70]. ... Enzyme immobilized nanomaterials as electrochemical biosensors for detection of biomolecules Article May 2022 ENZYME MICROB TECH Meena Nemiwal Tian C. Zhang Dinesh Kumar Biosensors have emerged as a potential tool for selective and sensitive detection. Biomolecules like enzymes, deoxyribonucleic acid (DNA), and antibodies are used as recognition sites in biosensors due to their high selectivity, sensitivity, and signal-to-noise ratio. Nowadays, biosensors are used to detect many vital biomolecules such as glucose, urea, and cholesterol. Exploiting enzymes for the selective detection of target molecules are very good but may not be easily accomplished as enzymes often need to be immobilized onto support materials, which requests either modifying the surface or using appropriate linkers to synthesize biosensors. Currently, many biosensors’ signal strength and stability still are low, which encourages the fabrication of functionalized nanomaterials/nanoparticles to enhance electrochemical performance. The review covers current progress in modifying enzyme immobilized biosensors to detect biologically essential molecules (glucose, urea, cholesterol). A discussion of the different types of enzyme immobilization is also provided. This review may be helpful for researchers to build rational and novel biosensors for efficient enzyme immobilization and sensing applications. View Show abstract ... These nanomaterials have gained attention because of their excellent photoluminescence yields and high photostability, which make them comparable to traditional quantum dots, but less toxic for biological systems [19,23,24]. C-Dots are attractive for their application as fluorescent probes for bioimaging [25,26], drug delivery [27][28][29], and optical biosensors development for environmental [30,31], agroindustry [32,33], and public health [34][35][36][37], with detection limits on nM order [38] [39][40]. The obtention of different types of C-Dots depends on the original feedstock, the physicochemical characteristics of the feedstock, synthesis conditions [23] and the doping with heteroatoms and surface modifications [41]. ... Synthesis, Characterization and Ecotoxicity Evaluation of Biochar-Derived Carbon Dots from Spruce Tree, Purple Moor-Grass and African Oil Palm Article Full-text available Jun 2021 Kaory Barrientos Maria Gaviria Juan Pablo Arango Marisol Jaramillo Biochar-derived C-Dots from Picea, Molinia caerulea and Elaeis guineensis were synthesized through a hydrothermal process, and their physicochemical and optical characteristics and environmental effects were compared. These C-Dots were characterized by techniques such as Attenuated Total Reflection–Fourier Transform Infrared (ATR-FTIR), UV-Vis spectrophotometry, fluorescence spectroscopy, dynamic light scattering (DLS), Z potential, and High-Resolution Transmission Electronical Microscopy (HR-TEM). The ecotoxicity tests were performed using the Microtox™ test, making this study one of the few that use this method. The C-Dots from Molinia caerulea showed the best quantum yield (QY) of 8.39% and moderate ecotoxicity, while Elaeis guineensis has the lowest QY (2.31%) but with zero toxicity. Furthermore, the C-Dots from Picea presents good optical properties but showed high toxicity and limits its use. Finally, all C-Dots showed functional groups that could be biofunctionalized with biomolecules, especially C-Dots from Molinia caerulea and Elaeis guineensis show potential for use in the development of optical biosensors. View Show abstract ... The cuproprotein enzyme Tyr was chosen for its ability to catalyze the oxidation of phenolic compounds, such as BPA. Furthermore, Tyr has higher specificity toward BPA than other polyphenol oxidases [25] [26] [27]. Figure 1a,b show the scanning electron microscopy images of PA6/PAH and PA6/ PAH@AuNPs nanofibers, respectively. Prior to the functionalization step, the PA6/PAH nanofibers ( Figure 1a) showed a smooth and bead-free surface and an average diameter of 88 ± 24 nm. ... Electrochemical Detection of Bisphenol A by Tyrosinase Immobilized on Electrospun Nanofibers Decorated with Gold Nanoparticles Article Full-text available Jan 2021 Luiza A. Mercante Leonardo E O Iwaki Vanessa P. Scagion Daniel S Correa Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) employed in industrial processes that causes adverse effects on the environment and human health. Sensitive and inexpensive methods to detect BPA are therefore needed. In this paper, we describe an electrochemical biosensor for detecting low levels of BPA using polymeric electrospun nanofibers of polyamide 6 (PA6) and poly(allylamine hydrochloride) (PAH) decorated with gold nanoparticles (AuNPs), namely, PA6/PAH@AuNPs, which were deposited onto a fluorine-doped tin oxide (FTO) substrate. The hybrid layer was excellent for the immobilization of tyrosinase (Tyr), which allowed an amperometric detection of BPA with a limit of detection of 0.011 μM in the concentration range from 0.05 to 20 μM. Detection was also possible in real water samples with recoveries in the range of 92–105%. The improved sensing performance is attributed to the combined effect of the large surface area and porosity of PA6/PAH nanofibers, the catalytic activity of AuNPs, and oxidoreductase ability of Tyr. These results provide a route for novel biosensing architectures to monitor BPA and other EDCs in water resources. View Show abstract ... Nanobiochars have also been applied to adsorb volatile organic compounds and for contaminant sensing applications (Dong et al., 2018b; He et al., 2020; Xiang et al., 2020). Hickory wood nanobiochar made from ball-milling biochars prepared at 300, 450, and 600 C, were used to capture the volatile organic compounds acetone, ethanol, chloroform, cyclohexane, and toluene (Xiang et al., 2020). ... Nanobiochar: A sustainable solution for agricultural and environmental applications Chapter Dec 2020 Tej Pratap Manvendra Patel Charles U Pittman Dinesh Mohan Biochar, a carbonaceous product of lignocellulosic biomass pyrolysis, has been shown to have numerous applications. Water remediation, contaminant immobilization, nutrient retention, soil fertility enhancements, and plant growth improvements are important biochar applications. However, modifications of biochar can change its properties and thus provide enhancements in biochar applications. Reducing biochar’s size to the nanolength scale to make “nanobiochar” gives a high surface area, high porosity, increased surface functional groups, and surface active sites. Increased contaminant adsorption, enhanced nutrient retention, and contaminant sensing are important applications of nanobiochar. This chapter summarizes the development, characterization, and applications of nanobiochar. Various nanobiochar development techniques with a special focus on ball-milling have been provided. Nanobiochar characterization techniques and its properties as compared to biochar have also been summarized. Nanobiochar applications in contaminant removal, agricultural applications, and sensing were also summarized. View Show abstract ... The chemical co-precipitation means that the BC is dispersed in the magnetic precursor solution, and by dropping the alkaline solution, the magnetic precursor generates magnetic precipitation on the surface of the BC (Fig. 2). The MBC obtained by this method is relatively stable and the operation process is easy to control, but the addition of alkali will increase the cost (Alchouron et al., 2020Bhushan et al., 2020 He et al., 2020b Tong et al., 2020. The magnetic precursor commonly used in chemical co-precipitation is Fe 3+ and Fe 2+ with a molar ratio of 2:1, and the reaction in the preparation process is as follows (Wu et al., 2018): ... Preparation of magnetic biochar and its application in catalytic degradation of organic pollutants: A review Article Apr 2021 SCI TOTAL ENVIRON Zhuqing Feng Rongfang Yuan Fei Wang Huilun Chen In recent years, magnetic biochar (MBC) has been greatly concerned because of its magnetic separation characteristics, and has been successfully used as a catalyst in the catalytic degradation of organic pollutants. However, there is currently a lack of a more systematic summary of MBC preparation methods, and no detailed overview of the catalytic mechanism of MBC catalysts for the degradation of organic pollutants. Therefore, we carry out this work to fill the above gaps. At first, we summarize the raw materials, preparation methods, and types of MBC in detail, and emphasize the MBC prepared by iron-containing sludge. Then, the catalytic mechanisms of MBC in peroxydisulfate, peroxymonosulfate, Fenton-like, photocatalysis, and NaBH4 systems are carefully summarized, highlighting the contribution of various parts of MBC in catalysis. The degradation efficiency of organic pollutants in the above systems is evaluated. Finally, the stability and reusability of MBC catalysts are evaluated. In conclusion, this review contributes a meager force to the future development of MBC. View Show abstract ... showing both high sensitivity and outstanding electrochemical activity 36 . ... Nanobiochar: Production, Properties, and Multifunctional Applications Article Aug 2020 Sammani Ramanayaka Meththika Vithanage Daniel Alessi Yong Sik Ok Nanobiochar has received much recent attention among engineered biochars owing to its useful chemical and physical properties. Research efforts have attempted to discover novel methods for nanobiochar preparation and applications. In this review, we summarize all the reported literature on various aspects of nanobiochar preparation, production and use. Often, bulk parent biochar obtained from biomass pyrolysis, mechanically ground using different milling processes to fabricate nanobiochar. Apart from mechanical means, direct fabrication of nanobiochar through flash heating resulting in graphitic nanosheets have been reported. Process conditions applied to the parent biochar directly influence the properties of the resulting nanobiochar. For instance, over 70% out of 33 nanobiochars derived from biomass pyrolyzed above 450 °C demonstrated 32 times greater BET specific surface areas than nanobiochars produced at <450 °C. Nanobiochar has diverse applications, such as wastewater treatment, health care applications, use as an electrode material, and in supercapacitors and sensors, owing to its wide range of physical and chemical properties. However, the toxicity of nanobiochar to human and ecosystem health has not received sufficient research attention. More research should be performed to elucidate the drawbacks, such as high agglomeration potential and low yield, of nanobiochar for practical uses. Furthermore, reported data is insufficient to obtain a clear idea of the nature and behavior of nanobiochar, despite growing interest in the research topic. Hence, future research should be driven towards exploring techniques to improve the yield of nanobiochar, reduce agglomeration, upscale it for electrode supercapacitor production and understanding toxicological aspects. View Show abstract ... Tan et al., 2015). Recently, it was demonstrated the biochar would have good electrochemical property by controlling the pyrolysis conditions during preparation and have the potential to be applied for electrode construction (Dong et al., 2018a; He et al., 2020) . By thinking of easy synthesis and extremely low-cost properties of biochar, it would be constructive and meaningful to newly investigate on the feasibility of biochar for the preparation of advanced electrochemical sensor such as the paper immunosensor in this research. ... Nanobiochar paper based electrochemical immunosensor for fast and ultrasensitive detection of microcystin-LR Article Aug 2020 SCI TOTAL ENVIRON Lan Yao Lingzhi He Yuesuo Yang Yunxian Piao A portable, cheap and sensitive paper type electrochemical immunosensor was developed with conductive nanobiochar paper as the conductive layer and utilized for sensitive detection of microcystin-LR (MCLR) toxin in water. The paper immunosensor was constructed by coating of highly conductive and dispersible nanobiochar particle (nBC) and anti-MCLR antibody on the filter paper via dipping-drying method. The presence of MCLR could be specifically quantified amperometrically by the nBC-paper immunosensor with the response time of less than 5 min, and the lowest detection limit of 17 pM (0.017 μg/L) was achieved. Moreover, the proposed immunosensor exhibited high selectivity, reproducibility and storage stability, and was also used for environmental water detection with satisfactory recovery. The successful fabrication of low cost and ubiquitous biochar based paper type electrochemical immunosensing system would have significant value for the development of highly cost-effective electrochemical device. View Show abstract ... Magnetic NPs can be used with organic and inorganic materials to immobilize biomolecules [111,113], as in the example of Tyr immobilized on GCE with biochar nanoparticles (BCNPs) functionalized with Fe 3 O 4 nanoparticles (MNPs) and carboxylic functional groups (-COOH) [114] . This magnetic-assisted electrochemical biosensor was capable of detecting BPA at nanomolar levels in spiked water samples. ... Polyphenol oxidase-based electrochemical biosensors: A review Article Jul 2020 ANAL CHIM ACTA Paulo Raymundo-Pereira Tiago Almeida Silva Fabio Caetano Orlando Fatibello-Filho The detection of phenolic compounds is relevant not only for their possible benefits to human health but also for their role as chemical pollutants, including as endocrine disruptors. The required monitoring of such compounds on-site or in field analysis can be performed with electrochemical biosensors made with polyphenol oxidases (PPO). In this review, we describe biosensors containing the oxidases tyrosinase and laccase, in addition to crude extracts and tissues from plants as enzyme sources. From the survey in the literature, we found that significant advances to obtain sensitive, robust biosensors arise from the synergy reached with a diversity of nanomaterials employed in the matrix. These nanomaterials are mostly metallic nanoparticles and carbon nanostructures, which offer a suitable environment to preserve the activity of the enzymes and enhance electron transport. Besides presenting a summary of contributions to electrochemical biosensors containing PPOs in the last five years, we discuss the trends and challenges to take these biosensors to the market, especially for biomedical applications. View Show abstract ... Compared with other BPA removal methods, bio-enzymatic methods have attracted increasing attention in wastewater treatment due to their mild treatment conditions and efficient reaction efficiency (He et al., 2020) . Among various enzymes, laccase has attracted much attention in the degradation of pollutants due to its high efficiency and substrate diversity (Taghizadeh et al., 2019). ... Construction of luffa sponge-based magnetic carbon nanocarriers for laccase immobilizatio and its application in the removal of bisphenol A Article Feb 2020 BIORESOURCE TECHNOL Chengyu Zhang Shengping You Yudong Liu Zhonghe He View Physical Activation and Nanoscale Transformation of Biochar Using Different Mechanochemical Techniques Chapter Jul 2023 Biochar is a solid carbonaceous pyrolyzed product prepared from various lignocellulosic biomass (agriculture and crop residues), sewage sludge, waste products obtained from different sources, litters, animal manures, etc., under limited oxygen conditions. In recent times, biochar applications in soil remediation, removal of hazardous organic and inorganic contaminants, wastewater treatment, electrode material, catalysis, etc., have drawn worldwide curiosity because of its unique properties. Instead of having unique properties, biochar application range is still limited due to its heterogeneous surface functionality, low porosity, and less adsorption capacity. The physicochemical properties of carbonaceous materials can be improved through various physical and chemical techniques, and biochar may be a better carbonaceous material due to its inexpensive and easy production process. Among physical modification techniques, steam activation is an environmentally and economically sustainable easy modification technique free from the use of any chemicals. With the advancement of nanotechnology, nanoscale engineering of biochar has captured care from scientific societies due to its nano-sized fabrication with excellent physical, chemical, and surface properties. Various mechanochemical techniques such as ball milling, microwave irradiation, and magnetic modification are vastly used for the nanoscale fabrication of biochar materials. Nanoscale engineering further increases the stability and efficiency of raw biochar material over physical modification techniques. This chapter includes steam activation used for physical activation of biochar as well as three selected mechanochemical techniques, viz. ball milling, microwave irradiation, and magnetic modification used for nanoscale modification of biochar along with their application in various fields. Biochar modification and its fabrication into nanoscale form is not only a novel approach but also provides a more advanced and efficient waste management technique.KeywordsBall millingMagnetic modificationsMicrowave irradiationModification of biocharNano-biochar materialsSteam activation View Show abstract Hydrothermally derived reduced and hierarchically porous biochar for rapid and sensitive electrochemical detection of benzo[a]pyrene Article Jun 2023 ELECTROCHIM ACTA Xinyao Wang Dongpeng Mao Penghu Duan Yunxian Piao View Recent Advances in Nano-metal Oxide-Biochar Composites for Efficient Removal of Environmental Contaminants Article Apr 2023 REV ENVIRON CONTAM T Akshay Kumar Dey Md. Ahmaruzzaman Along with global industrialization and rapid urbanization, the perpetual conception of sustainable environmental remediation is to be delivered as its utmost priority. For many years, metal oxide-biochar has followed biochar in the removal of harmful pollutants from natural resources and issues. The relatively lower effectiveness and efficiency of standard biochar (BCs) and metal biochar composites (MBCs) result in the continuation of further research efforts, as nano-metal oxide biochar composites and nano-metal–metal oxide biochar composites (NMOBCs) have emerged as environmental remediation pioneers in recent days. The novel NMOBCs have more flexible physicochemical properties and enhance the removal efficiency of environmental contaminants through adsorption and photocatalytic degradation. The absence of a complete description of adsorption and a systematic summary of NMOBC catalytic processes is still being investigated. Therefore, in this review, efforts have been made to fill these gaps to some extent by emphasizing synthetic methods, properties, and characterization of raw materials, a comprehensive overview of adsorptive properties, and a systematic summary of the catalytic activities of novel NMOBCs. Also, the meager challenges and recent developments up to the mark are discussed. Further, suggestions and recommendations for the modifications of physicochemical properties of NMOBCs are proposed to make them potentially applicable in the enhanced adsorption and photocatalytic degradation of various inorganic and organic environmental contaminants. View Show abstract Recent advances in nanomaterials based sustainable approaches for mitigation of emerging organic pollutants Article Feb 2023 CHEMOSPHERE Monika Bhattu Jagpreet Singh Emerging pollutants (EPs) are a category of pollutants that are relatively new to the environment and recently garnered a lot of attention. The majority of EPs includes endocrine-disrupting chemicals (EDCs), antibiotic resistance genes (ARGs), and pharmaceutical and personal care products (PPCPs). Exposure to contaminated water has been linked to an increase in incidences of malnutrition, intrauterine growth retardation, respiratory illnesses, liver malfunctions, eye and skin diseases, and fatalities. Consequently, there is a critical need for wastewater remediation technologies which are effective, reliable, and economical. Conventional wastewater treatment methods have several shortcomings that can be addressed with the help of nanotechnology. Unique characteristics of nanomaterials (NMs) make them intriguing and efficient alternative in wastewater treatment strategies. This review emphasis on the occurrence of divers emerging organic pollutants (EOPs) in water and their effective elimination via different NMs based methods with in-depth mechanisms. Furthermore, it also delves the toxicity assessment of NMs and critical challenges, which are crucial steps for practical implementations. View Show abstract Biochar: An environmentally friendly platform for construction of a SARS-CoV-2 electrochemical immunosensor Article Nov 2022 SCI TOTAL ENVIRON Marcia Gabriela Pianaro Valenga Gustavo Martins Thomas A. C. Martins Márcio F Bergamini Waste management is a key feature to ensure sustainable consumption and production patterns, and to combat the impacts of climate change. In this scenario, the production of biochar from different biomasses results in environmental and economic advantages. In this study, biochar was produced from sugarcane bagasse pyrolysis, to immobilize biomolecules, in order to assemble an electrochemical immunosensor to detect antibodies against SARS-CoV-2. For this, screen-printed carbon electrodes (SPCE) were modified with a dispersion of biochar and used to immobilize the receptor-binding-domain (RBD) against virus S-protein, through EDC/NHS crosslinking reaction. Under the best set of experimental conditions, negative and positive serum samples responses distinguished based on a cutoff value of 82.3 %, at a 95 % confidence level. The immunosensor showed selective behavior to antibodies against yellow fever and its performance was stable up to 7 days of storage. Therefore, biochar yielded from sugarcane bagasse is an ecofriendly material that can be used as a platform to immobilize biomolecules for construction of electrochemical biosensors. View Show abstract The Electrochemical Detection of Dopamine Based on the Cobalt-Modified Nitrogen Doping Carbon Aerogels from Biomass Article Aug 2022 Xun Zou Yuying Chen Xiangfei Song Zhao Peng The sensitive detection of dopamine (DA), especially the accurate monitoring of cell secreted DA, is necessary to the study of pathogenesis of central nervous system disease. Here, the cobalt-modified nitrogen doping carbon aerogels (Co-NCA) with three-dimensional pores were successfully synthesized through the hydrothermal and freeze-drying method of the biomass. As a natural substance, starch is the carbon source of Co-NCA, which is environmentally friendly. The electrochemically active area of an electrode modified by Co-NCA was about 1.53 times that of carbon aerogels. Benefiting from the Co and N element, the Co-NCA modified electrode (Co-NCA/GCE) showed that Co-NCA had a wider linear range (0.2-200 and 200-1000 μM) and a lower detection limit (55.6 nM) towards DA. The reproducibility and repeatability of Co-NCA/GCE for detecting 500 M DA was 2.21% and 0.797%, respectively. After being stored at 4°C for 7 days, the current response of Co-NCA/GCE still maintained 96.5% of the initial current value. Finally, the Co-NCA/GCE was successfully applied to the monitoring of DA, which was released from PC12 cells after stimulated by 50 mM K+. View Show abstract Ultrasensitive and highly reusable electrochemical sensor with ion imprinted nanobiochar Article Aug 2022 SENSOR ACTUAT B-CHEM Dongpeng Mao Penghu Duan Jiafeng Hu Yunxian Piao A novel electrochemical sensor for the ultrasensitive monitoring of Pb²⁺ and Cd²⁺ was developed by fabrication of highly conductive and target selective electrode using ball milling biochar (BBC) featured with high conductivity and rich oxygenic functional groups as the conductive material, and cavities of ion-imprinted polymer as specific binding moieties. The nanosized BBC was synthesized with process of high temperature pyrolysis and rigorous ball milling. The ion imprinted BBC electrode was constructed by in situ electropolymerization of L-Cysteine and template metal ions on the BBC that was modified on glassy carbon, and subsequent removal of templates. The BBC presented great electron transfer ability, and contained increased oxygenic compounds than the mild ball milling prepared one, which attributed to formation of stable and cavity-rich imprinting polymer layer and enhancement of sensing signal. With the anodic dissolved differential pulse voltammetry, the electrode could detect extremely low levels of Pb²⁺ and Cd²⁺ with lowest detection limits of 5.86 fM and 0.883 aM, and the linear ranges were 25 fM ~ 1 µM and 0.1 fM ~ 1 µM, respectively. The electrode also presented anti-interference to other irrelevant ions and organic compounds, and could reuse at least seven times without decrease of sensing signal. View Show abstract Copper nanostructures anchored on renewable carbon as electrochemical platform for the detection of dopamine, fluoxetine and escitalopram Article Jun 2022 Clara M.B Trindade Martin Kássio Leme da Silva Ivana Cesarino For the first time, renewable carbon (RC) from bamboo-biomass modified with copper nanoparticles (CuNPs) has been prepared via a chemical reduction step for the electroanalytical detection of dopamine (DOP), fluoxetine (FXT) and escitalopram (EST). The synthesized RC-CuNPs nanocomposite morphology and structural composition were characterized by FEG-SEM along with EDS analysis. The electroanalytical behavior of the target analytes was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The analytical range of detection of DOP, FXT, and EST was 0.05 – 5.0, 0.1 – 10, and 0.02 – 5.0 µmol L⁻¹, respectively. The limits of detection (LOD) were found to be 0.04, 0.05, and 0.25 µmol L⁻¹, respectively. In addition, the standard addition method was used for the detection and recovery of DOP in synthetic human urine and EST in tap water samples. The synergistic effect of CuNPs coupled with the highly porous RC material provided a sensitive, low-cost, and easy to prepare sensing platform for detecting target analytes in clinical and environmental samples. View Show abstract Biomedical uses of Enzymes Immobilized by Nanoparticles Chapter Full-text available May 2022 Syed Raza Ali Naqvi, Noman Razzaq, Tahseen Abbas, Ameer Fawad Zahoor, Matloob Ahmad, Amjad Hussain, and Sadaf Ul Hassan Noman Razzaq Tahseen Abbas Sadaf ul Hassan Immobilized enzymes are now a significant and appropriate area of modern technologies. Immobilization of enzymes on nanoparticles (NPs) especially magnetic nanoparticles (MNPs) not only increase the stability of the enzymes by protecting the active site but also facilitates the separation mode. Immobilized technology is considered effective in context of running cost to exercise immobilized enzymes technique. Nowadays, variety of magnetic nanoparticles are available such as chitin-chitosan magnetic nanoparticles, Fe3O4 magnetic nanoparticles, bacteriophages T4 capsid novozym-435 etc. which are quite fit for loading enzymes and to use fruitfully. The main focus in this piece of work is that how immobilized enzymes are helpful in different biomedical uses and what kind of enzymes and nanoparticles could be hyphenated to take advantage in health care sectors. Different method of enzymes immobilization will also be discussed in details including both physical methods and chemical methods of loading enzymes on nanoparticles. View Show abstract Ultrasensitive electrochemical determination of bisphenol A in food samples based on a strategy for activity enhancement of enzyme: Layer-by-layer self-assembly of tyrosinase between two-dimensional porphyrin metal–organic framework nanofilms Article May 2022 CHEM ENG J Junping Ma Jinhang Yuan Yiying Xu Jianbin Zheng Bisphenol A, as an endocrine disruptor, has aroused widespread attention for its safety. Electrochemical enzyme-based biosensor is a powerful tool for the determination of bisphenol A, but the instability of enzyme limits its practical application. Assembling biomacromolecules in metal–organic frameworks (MOFs) can preserve their biological functions in harsh environments. In this work, tyrosinase (Tyr) was assembled layer-by-layer in an ultrathin copper–porphyrin MOF nanofilm ([email protected]–TCPP) via a simple one-step solvothermal method, and an ultrasensitive electrochemical biosensor was fabricated for bisphenol A detection. Compared with native Tyr or a traditional surface-adsorbed structure of Tyr on Cu–TCPP nanofilm, [email protected]–TCPP retained superior enzymatic activity when exposed to elevated temperatures and extreme acidity or basicity, the sensor exhibited significantly enhanced thermal and long-term storage stability, as well as acid/base tolerance. In addition, the electrochemical biosensor based on [email protected]–TCPP showed significantly enhanced sensing performance, including a wide linear range of 3.5 nM–18.9 μM, with a detection limit of 1.2 nM. For the first time, the fabrication of an electrochemical biosensor relying on enzyme assembled between two-dimensional MOF nanomaterial layers has been reported. This method greatly improves the activity and stability of the biosensor, and provides a promising strategy for promoting the application of enzyme biosensors in harsh detection environments, discovering and controlling environmental pollutants and food hazards. View Show abstract Immobilizing chromate reductase NfoR on magnetic biochar reduced Cr(VI) in copper-containing wastewater Article May 2022 J CLEAN PROD Huawen Han Peizhi Song Zhuoshan Cai Xiangkai Li Heavy metals (e.g. copper) in smelting wastewater inhibited microbial Cr(VI) reduction, previous report found that a novel chromate reductase NfoR exhibited Cu(II)-enhanced activity and broad-spectrum metal tolerance. In this study, magnetic biochar (MB) from pine sawdust was fabricated to immobilize NfoR via covalent binding. MB-NfoR showed a 2.6- and 2.1-folds higher activity in reducing Cr(VI) to Cr(III) compared to free NfoR and MB, respectively. XPS and EPR analysis showed that NfoR catalysis and persistent free radicals (PFRs) are responsible for Cr(VI) reduction mechanism of MB-NfoR. The addition of Cu(II) increased the MB-NfoR activity 2.3-fold compared to that of without Cu(II). The maximum Cr(VI) removal by MB-NfoR in the pure Cr(VI) solution was 98% at 45 °C and pH 6.0, and the MB-NfoR retained 68.3% of its initial activity even after five consecutive cycles. Application of MB-NfoR in smelting wastewater can simultaneously remove 94% Cr(VI) and 52.1% Cu(II). Furthermore, treated smelting wastewater by MB-NfoR showed no phytotoxicity and enriched numerous oligotrophic bacteria. This study provides a novel ‘one-stop’ strategy for decontamination of effluents combined with heavy metals, utilizing synergies offered by constituents in such waste streams. View Show abstract Bioremoval of toxic malachite green from water through simultaneous decolorization and degradation using laccase immobilized biochar Article Mar 2022 CHEMOSPHERE Deepshikha Pandey Achlesh Daverey Kasturi Dutta Kusum Arunachalam In this study, decolorization and degradation of malachite green dye was studied using the laccase immobilized pine needle biochar. Successful immobilization of biochar was achieved by adsorption and confirmed through scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX), Fourier transform infrared spectroscopy (FTIR). High laccase binding of 64.4 U/g and high immobilization yield of 78.1% was achieved using 4U of enzyme at pH3 and temperature 30 °C. The immobilized laccase retained >50% relative activity in the pH range 2–7, >45% relative activity at 65 °C and >55% relative activity at 4 °C for 4 weeks. The re-usability of immobilized enzyme was checked with 2, 2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) substrate and enzyme retained 53% of its activity after 6 cycles. Immobilized laccase was used for the degradation and decolorization of azo dye malachite green in aqueous solution. More than 85% removal of malachite green dye (50 mg/L) was observed within 5 h. FTIR and high performance liquid chromatography (HPLC) analysis clearly indicated the breakdown of dye and presence of metabolites (leuco malachite green, methanone, [4-(dimethyl amino)pheny]phenyl and 3-dimethyl-phenyl amine) in gas chromatography–mass spectrometry (GC-MS) analysis confirmed the dye degradation. Phytotoxicity analysis indicated that the enzymatic degradation resulted in lesser toxic metabolites than the original dye. Thus, laccase immobilized biochar can be used as an efficient biocatalytic agent to remove dye from water. View Show abstract A Novel Electrochemical Biosensor for Bisphenol A Detection Based on Engineered Escherichia coli Cells with a Surface-display of Tyrosinase Article Nov 2021 SENSOR ACTUAT B-CHEM Shuai Zhao Tuoyu Zhou Aman Khan Xiangkai Li Electrochemical enzyme biosensors based on various modified electrodes have been developed for bisphenol A detection. However, enzyme activity loss and high cost limit their application. In this study, an electrochemical biosensor was developed for bisphenol A detection by displaying tyrosinase on the surface of Escherichia coli cells, followed by adsorption onto a glassy-carbon electrode. The biosensor exhibited a linear relationship between the concentration range of 0.01-100 nM BPA (R² = 0.9958) and the current peak. The detection limit was 0.01 nM, which is higher than that of other chemically modified tyrosinase biosensors. For the detection of bisphenol A in tea and juice samples, the fabricated biosensor presented respectable accuracy comparable to that of high-performance liquid chromatography. Furthermore, high recovery rates (97.78%-100.32%) were obtained. Therefore, this biosensor could be a promising and reliable analytical tool for the detection of bisphenol A. View Show abstract Removal and sensing of emerging pollutants released from (micro)plastic degradation: Strategies based on boron-doped diamond electrodes Article Oct 2021 Thiago M.B.F. Oliveira Francisco Wirley Paulino Ribeiro Simone Morais Adriana Correia The negative impacts of microplastics (MPs) on the environment and human health cannot be unnoticed. Several classes of emerging pollutants with endocrine disrupting properties such as bisphenol A and its analogues, phthalates, among others, have been reported to migrate out of plastics entering the aquatic environment. Thus, this review aims to draw attention to the significant potential of the boron-doped diamond electrode (BDDE) to contribute to the implementation of mitigation actions for MPs pollutants. The latest studies in the two main fields of the use of the BDDE i.e., treatment of wastewater by electrochemical oxidation and as sensor for pollutants monitoring, are herein reviewed and their main findings highlighted. View Show abstract Recent advances in carbon nanomaterials-based electrochemical sensors for phenolic compounds detection Article Aug 2021 MICROCHEM J Shanshan Fu Yuan Zhu Yi Zhang Dongbo Wang Phenolic compounds (PCs) exist widely in natural environment and human life, which pose a huge threat to human health due to the toxic effects. Therefore, it is necessary to develop effective, robust, and reliable methods for PCs detection. Electrochemical sensors have been attracted widespread attention in detection due to their simple instruments, high sensitivity, fast response, and good selectivity. In addition, carbon nanomaterials (CNMs) have shown great application potential in electrochemical sensing technology due to their unique electrical, optical, thermal, mechanical and chemical properties. Therefore, in this review, the latest technologies of electrochemical sensors based on CNMs for PCs detection over the past decade (2010-2020) were introduced. The application of CNMs involving graphene and its derivatives, carbon nanotubes, graphitic carbon nitride, carbon dots, carbon nanofibers, carbon nanospheres and mesoporous carbon in electrochemical sensors were summarized. Furthermore, it also included the challenges and prospects of CNMs-based electrochemical sensors for PCs detection. View Show abstract Highly Selective Entrapment of His-Tagged Enzymes on Superparamagnetic Zirconium-Based MOFs with Robust Renewability to Enhance pH and Thermal Stability Article Jul 2021 Bin Wei Haichang Xu Leiyu Cheng Hao Liang View Hybrid magnetic nanoparticles for electrochemical biosensors Chapter Jan 2021 Anabel Villalonga Reynaldo Villalonga Diana Vilela Hybrid nanomaterials, defined as the combination of at least one nanomaterial with one or more other materials at the nanoscale, generating improved properties that the individual components do not possess, have been shown to be ideal platforms for the construction of highly performance electrochemical sensor and biosensors. Among them, hybrid magnetic nanoparticles (HMNP) have been broadly used in electroanalytical chemistry because they offer outstanding advantages in overcoming the conventional challenges that electrochemical biosensing presents. The greatness of these HMNP comes from the possibility of creatively tuning their properties by the combination of different materials according to the intended end use, which opens new horizons for the future design of better on-demand electrochemical biosensors. This chapter provides a general overview of the main achievements yielded in the use and integration of HMNP as preconcentration, labeling, transduction, and amplification elements for the development of electrochemical biosensing devices in the recent years. The chapter is structured in three main sections focused on the classification of HMNP, their synthesis, and their applications, all in the field of electrochemical biosensors. View Show abstract Applicative Insights on Nascent Role of Biochar Production, Tailoring and Immobilization in Enzyme Industry -A Review Article May 2021 PROCESS BIOCHEM Naviljyot Kaur Priyanka Bhardwaj Gursharan Singh Shailendra Kumar Arya Biochar or agrichar the carbon rich source has been appreciated globally for its versatile properties like large surface area, high level porosity, thermal stability and long shelf life. The advent of such properties and the fact that it can be obtained at low operational costs has created phenomenal applications for biochar to be used in biotechnological and agricultural industries. Availability and high cost of production of enzymes has lead immobilization to be served as fruitful technique to maintain the price efficacy. Endowing to its properties like surface availability of functional groups, being an exceptional biosorbent it is availed as potential carrier for immobilization. For better applicability, various physio-chemical tailoring strategies have been adopted to perk up the usage. The main mechanism that works for biochar immobilization is adsorption due to high surface porosity, surface area of biochar. Further aiding to perks of adsorption being simple, cost effective and economical approach that maintains structural integrity of the enzymes. In addition to this covalent binding can vanquish the demerit enzyme leeching caused by adsorption.This review centers on various methods for production of biochar, a range of tailoring mechanisms being followed, and applicative usages of biochar immobilized enzymes in diverse processes. View Show abstract Production, characterization, activation and environmental applications of engineered biochar: a review Article Jan 2021 Dilipkumar Akhil Divya Lakshmi Ashokkumar Kartik Gopinath Kannappan Panchamoorthy Increased industrial and domestic activities have induced pollution, global warming and resources depletion, calling for advanced remediation techniques. For instance, biochar synthesized from waste materials is an ecofriendly and low-cost material for the remediation of contaminants, yet raw biochar is poorly efficient. Therefore, biochar properties can be improved by ultrasonication, metal impregnation, surfactant modification, microwave, electrochemical and plasma treatment techniques. Biochar properties include adsorption capacity, active surface area, uniform distribution of pores, chemical attributes and mechanical stability. Engineered biochar thus exhibits high contaminant removal efficiency of 90–99%, tolerance to wide range of pH of 3–9 and high specific surface area of more than 400 cm3/g. For example, modified cornstalk biochar displays mercury adsorption capacity of 269.4 mg/g. The usage of magnetic biochar allows a β-estradiol adsorption capacity of 98.8 mg/g. Engineered biochar can act as an ecofriendly soil amendment and a highly efficient tool to recover contaminants from water systems. View Show abstract Sugarcane biochar as novel catalyst for highly efficient oxidative removal of organic compounds in water Article Feb 2021 CHEM ENG J Zairan Liu Jiafeng Hu Lingzhi He Yunxian Piao Cost effective and environmental friendly biochar catalyst (SugBC900) with high catalytic activity toward peroxymonosulfate (PMS) was prepared by simple pyrolysis of sugarcane waste, and utilized for efficient degradation of organic pollutants. Taking advantages of high sp² hybrid carbon content, large surface area and excellent electron transfer ability, the SugBC900 coupled with PMS presented far greater catalytic degradation ability toward bisphenol A (BPA) as model pollutant when compared to the SugBC700, SugBC800 and other carbonaceous catalysts. The oxidative species of ¹O2 and O2•− were demonstrated to be dominant for catalytic degradation, and the role of SO4•− and •OH was minor. 20 mg/mL of BPA could be completely degraded with less than 60 min at pHs of 5.0–11.0, and at temperatures of 25–45 °C. And at the extremely acidic (pH of 1.0) or low temperature (10 °C) conditions, the degradation efficiencies could still reach more than 90%. The SugBC900 was also successfully utilized for the efficiently catalytic degradation of BPA and other organic pollutants in various real environmental water. Because of simple preparation by impregnating none of external elements, extremely low cost property, and robustness characteristic by having broad working conditions, the SugBC900 would have potential to be a good alternative to the conventional catalysts. View Show abstract In Situ-Forming Magnetic Fe 3 O 4 Nanoroses on Defect-Controllable Mesoporous Graphene Oxide for Enzyme-Mimic Sensing Article Oct 2020 Xiaoyue Yao Tao Liu Ying Xie Wanqin Jin View Uniform manganese-loaded titanium dioxide nanotube arrays for accurate detection of trace Cd2+ in water, soil and tea: Enhanced stability and sensitivity Article Jun 2020 CHEM ENG J Min Jiang Ming-jun Ma Chu-Hong Lin Xing-Jiu Huang The poor stability of the signal obtained on nonuniform substrate, which results from the obvious difference in the size of ablation spots, the uneven distribution of enriched analytes, and the severe matrix effect, seriously affecting the practical application of electrochemical preconcentration supported laser-induced breakdown spectroscopy (ECP-LIBS). Here, self-organizing and ordered manganese loaded titanium dioxide nanotube arrays (Mn-TiO2 NTAs) on a titanium foil was employed as the working electrode to overcome the problem. The Mn-TiO2 NTAs were of equal height, hollow-structured and evenly arranged on the electrode, leading to uniform and shallow ablation spots after Gaussian laser irradiation (proven by MATLAB simulation), which reduced the matrix effect. As a result, a high sensitivity of 13347.7 counts μM⁻¹and low LOD of 0.01 μM for Cd²⁺ were achieved. Most importantly, the self-organizing and ordered Mn-TiO2 NTAs electrode exhibited outstanding stability and reproducibility (relative standard deviation, RSD, was less than 5%), while the Mn-TiO2 electrode modified through the drip coating method showed poor stability (RSD was greater than 20%). The proposed method perfectly achieved the anti-interference and accurate detection of Cd²⁺ in water, soil and tea. View Show abstract Current trends on turning biomass wastes into carbon materials for electrochemical sensing and rechargeable battery applications Article Jun 2020 Alessandro Pistone Claudia Espro The need to both avoid waste and develop sustainable technologies based on new renewable resources, has led to an encouraging research opportunity, focusing attention on the relative merits of applying different dedicated waste biomass as renewable, cheap, and abundant resources to minimize energy and material feedstock costs. Carbonaceous materials obtained from waste biomass have received comprehensive interest over the past years due to their attractive electronic and structural properties, which allow them as advanced materials for a wide variety of applications. Here, we highlight the range of waste biomass precursors available and current trends on bio-carbon production for its use in electrochemical devices for sensing systems and rechargeable batteries, which are expected to have a rapid scientific growing and industrial interest in the next years. View Show abstract Electrochemical Enzyme Biosensor Bearing Biochar Nanoparticle as Signal Enhancer for Bisphenol A Detection in Water Article Full-text available Apr 2019 SENSORS-BASEL Lan Yao Yang Liu Lingzhi He Yunxian Piao An electrochemical tyrosinase enzyme (Tyr) biosensor using a highly conductive sugarcane derived biochar nanoparticle (BCNP) as a transducer and signal enhancer (BCNPs/Tyr/Nafion/GCE) was developed for the sensitive detection of bisphenol A (BPA). The BCNPs/Tyr/Nafion/GCE biosensor exhibited improved amperometric current responses such as higher sensing signal, decreased impedance and lowered reduction potential compared with the Tyr/Nafion/GCE due to high conductivity property of the biochar nanoparticle. Under the optimized conditions, it could detect BPA in good sensitivity with linear range from 0.02 to 10 μM, and a lowest detection limit of 3.18 nM. Moreover, it showed a low Km value, high reproducibility and good selectivity over other reagents, and the BCNPs/Tyr complex solution also showed good stability with 86.9% of sensing signal maintained after one month storage. The biosensor was also successfully utilized for real water detection with high accuracy as validated by high performance liquid chromatography. Therefore, the biochar nanoparticle based enzyme biosensor proved to be a potential and reliable method for high performance detection of pollutants in the environment. View Show abstract Biosensor Array of Interpenetrating Polymer Network with Photonic Film Templated from Reactive Cholesteric Liquid Crystal and Enzyme‐Immobilized Hydrogel Polymer Article Full-text available May 2018 ADV FUNCT MATER Kyung Gyu Noh Soo Young Park A biosensor array is fabricated using an interpenetrating polymer network consisting of photonic film templated from reactive cholesteric liquid crystal (CLC) and enzyme-immobilized polyacrylic acid (PAA). The solid-state photonic film on the glass substrate is successfully templated by ultraviolet (UV) curing of the reactive CLC mixture of a reactive mesogen mixture of RMM 727 (from Merck) and a nonreactive chiral dopant of (S)-4-cyano-4′-(2-methylbutyl)biphenyl following the extraction of the chiral dopant. The acrylic acid monomer mixed with a cross-linker of tri(propylene glycol) diacrylate is infiltrated into the extracted space of the photonic film, and UV-cured with a photomask to obtain a patterned array-dot film. The interpenetrated cholesteric liquid crystal/hydrogel polymer network (CLC-hydrogel-IPN) array is further functionalized in the individual dots with urease, for a model study of biosensor array applications. The dots of the CLC-hydrogel-IPN array respond independently to the urea by a color change with high sensitivity and stability. Thus, the patterned CLC-hydrogel-IPN can be used as a new biosensor array for cost-effective and easy visual detection without any sophisticated instruments. View Show abstract Amperometric enzymatic determination of bisphenol A using an ITO electrode modified with reduced graphene oxide and Mn3O4 nanoparticles in a chitosan matrix Article Full-text available Jun 2017 MICROCHIM ACTA Md Azahar Ali Manish K Singh K.Kamil Reza Ashok Manikrao Biradar The authors describe a nanostructured hybrid platform for amperometic determination of the plasticizer bisphenol A (BPA). It consists of two dimensional sheets of reduced graphene oxide (rGO) that are decorated with Mn3O4 nanoparticles. The enzyme tyrosinase was immobilized on the surface of the hybrid nanosheets placed on an ITO electrode. The larger surface area and enhanced charge transfer properties of the rGO/Mn3O4 nanosheets provide a synergistic effect that results in excellent sensing performance including fast electron transfer kinetics, high electroactivity, good reproducibility and selectivity compared to other electrodes of that kind. Figures of merit include a sensitivity of 93.2 μA⋅nM⁻¹⋅cm⁻², an analytical range that extends from 0.01 to 100 μM, and a 10 nM lower detection limit. Its long-term stability of 8 weeks suggests the use of such enzyme electrodes as potential tools in clinical diagnostics. Graphical abstractSchematic of the fabrication of graphene based hybrid electrode using reduced graphene oxide and colloidal Mn3O4 nanostructures in presence of chitosan polymer. Further we demonstrate the electrochemical assay of the hybrid electrode for Bisphenol A detection. View Show abstract Cadmium removal potential by rice straw-derived magnetic biochar Article Full-text available Apr 2017 CLEAN TECHNOL ENVIR Zhongxin Tan Yuanhang Wang Alfreda Kasiulienė Ping Ai Cadmium removal from wastewater is a difficult problem, so our aim was to develop a novel biochar and method of biochar preparation for the high-efficiency removal of cadmium. First, common biochars created using different pyrolysis conditions were tested to determine the best absorbance using the iodine absorption test. Magnetic biochar was prepared using a one-step synthesis by treating rice straw with Fe²⁺/Fe³⁺ and pyrolizing using the working conditions that exhibited the best absorbance. Second, the prepared biochars were used as adsorbents for Cd(II) removal from a solution. The magnetic biochar showed high efficiency for the removal of Cd(II): up to 91 %. This rate is much greater than that of the original biochar. View Show abstract Biochar as a sustainable electrode material for electricity production in microbial fuel cells Article Full-text available Apr 2014 BIORESOURCE TECHNOL Tyler Huggins Heming Wang Joshua Kearns Zhiyong Jason Ren Wood-based biochars were used as microbial fuel cell electrodes to significantly reduce cost and carbon footprint. The biochar was made using forestry residue (BCc) and compressed milling residue (BCp). Side-by-side comparison show the specific area of BCp (469.9 m2 g−1) and BCc (428.6 cm2 g−1) is lower than granular activated carbon (GAC) (1247.8 m2 g−1) but higher than graphite granule (GG) (0.44 m2 g−1). Both biochars showed power outputs of 532 ± 18 mW m−2 (BCp) and 457 ± 20 mW m−2 (BCc), comparable with GAC (674 ± 10 mW m−2) and GG (566 ± 5 mW m−2). However, lower material expenses made their power output cost 17–35 US$ W−1, 90% cheaper than GAC (402 US$ W−1) or GG (392 US$ W−1). Biochar from waste also reduced the energy and carbon footprint associated with electrode manufacturing and the disposal of which could have additional agronomic benefits. View Show abstract Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry Article Full-text available Nov 2013 ANALYST Elisabetta Maiolini Elida Nora Ferri Agata Laura Pitasi Stefano Girotti Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays. View Show abstract Acidic and alkaline pretreatments of activated carbon and their effects on the performance of air-cathodes in microbial fuel cells Article Full-text available Jul 2013 Xin Wang Ningshengjie Gao Qixing Zhou Yujie Feng Activated carbon (AC) is a high performing and cost effective catalyst for oxygen reduction reactions (ORRs) of air-cathodes in microbial fuel cells (MFCs). Acidic (HNO3) and alkaline (KOH) pretreatments on AC at low temperature (85°C) are conducted to enhance the performance of MFCs. The alkaline pretreatment increased the power density by 16% from 804±70 to 957±31mWm(-2), possibly due to the decrease of ohmic resistance (from 20.58 to 19.20Ω) and the increase of ORR activities provided by the adsorbed hydroxide ion and extra micropore area/volume after alkaline pretreatment. However, acidic pretreatment decreased the power output to 537±36mWm(-2), which can be mainly attributed to the corrosion by adsorbed proton at the interface of AC powder and stainless steel mesh and the decreased pore area. View Show abstract Amperometric biosensor based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine-silk fibroin film and its application to determine bisphenol A Article Full-text available Feb 2010 Huanshun Yin Yunlei Zhou Jing Xu Lu-Sheng Zhu An amperometric bisphenol A (BPA) biosensor was fabricated by immobilizing tyrosinase on multiwalled carbon nanotubes (MWNTs)-cobalt phthalocyanine (CoPc)-silk fibroin (SF) composite modified glassy carbon electrode (GCE). In MWNTs-CoPc-SF composite film, SF provided a biocompatible microenvironment for the tyrosinase to retain its bioactivity, MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate and CoPc showed good electrocatalytic activity to electrooxidation of BPA. The cyclic voltammogram of BPA at this biosensor exhibited a well defined anodic peak at 0.625 V. Compared with bare GCE, the oxidation signal of BPA significantly increased; therefore, this oxidation signal was used to determine BPA. The effect factors were optimized and the electrochemical parameters were calculated. The possible oxidation mechanism was also discussed. Under optimum conditions, the oxidation current was proportional to BPA concentration in the range from 5.0 x 10(-8) to 3.0 x 10(-6) M with correlation coefficient of 0.9979 and detection limit of 3.0 x 10(-8) M (S/N=3). The proposed method was successfully applied to determine BPA in plastic products and the recovery was in the range from 95.36% to 104.39%. View Show abstract Activation mechanism of peroxymonosulfate by biochar for catalytic degradation of 1,4-dioxane: Important role of biochar defect structures Article Mar 2019 CHEM ENG J Da Ouyang Yun Chen Jingchun Yan Mengfang Chen Carbonaceous materials have been emerged as promising metal-free catalysts to activate peroxymonosulfate (PMS) for the degradation of organic contaminants. In the present study, a low-cost carbonaceous material, pine needle biochar was prepared under the oxygen limited pyrolyzation process and applied to activate PMS for the catalytic degradation of potentially carcinogenic 1,4-dioxane. Based on the batch experiments, the pyrolysis temperature has a significant influence on the potency of biochar to activate PMS. Within the biochar/PMS system, the degradation efficiencies of 1,4-dioxane were increased from 4.1 to 84.2% when the biochar pyrolysis temperatures were increased from 300 °C to 800 °C. Electron paramagnetic resonance (EPR) study and the quenching experiment verified that the dominant free radical within the system of biochar/PMS was [rad]OH. The EPR combined X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and Raman analysis implied that the defect of biochar contributed to the generation of SO 4[rad]− and [rad]OH and high pyrolysis temperatures would eliminate the excess of oxygen functional groups in biochar, which could modulate and transform sp ³ carbons, generating more defect structures, therefore, enhancing the activation potency of biochar towards PMS. Simultaneously, five major intermediates were identified and three possible degradation pathways were proposed in this study. In addition, 71.4% and 57.5% of 1,4-dioxane removal rates were achieved in 1,4-dioxane contaminated tap water and groundwater, indicating that biochar activating PMS is a promising technique for the remediation of 1,4-dioxane contaminated water. View Show abstract Hyphenation of enzyme/graphene oxide-ionic liquid/glassy carbon biosensors with anodic differential pulse stripping voltammetry for reliable determination of choline and acetylcholine in human serum Article Mar 2019 TALANTA Hassan M. Albishri Deia Abd El-Hady Acetylcholine (ACh) and its precursor choline (Ch) play important roles in many biological processes. It is expected that Alzheimer's disease is occurred due to the reduction in synthesis of ACh. On the other hand, the increase in the level of ACh results in a depression of heart rate and over production of saliva. Therefore, the quantitative determination of Ch and ACh is very important in biological media. In the current work, sensitive and selective biosensors composed of choline oxidase (ChO) and/or acetylcholine esterase (AChE) on graphene oxide-ionic liquid (GO-IL)/ glassy carbon electrode (GCE) hyphenated with anodic differential pulse stripping voltammetry (ADPSV) were firstly established for the determination of ACh and Ch in human serum samples. The molecular bond of ionic liquid 1-allyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (AMIM TFSI) with GO was investigated by FT-IR and UV–Vis techniques. Furthermore, the surface topography of ChO/GO–IL and AChE-ChO/GO–IL composites was investigated by SEM and XRD. Then, the electron transfer features of biosensors ChO/GO-IL/GCE and AChE-ChO/GO-IL/GCE were characterized by the electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The ADPSV was further used for the determination of Ch and ACh. The experimental parameters such as differential pulse working potential, differential pulse scan rate, equilibrium time and long-term stability were further optimized. Detection limits of 0.885 and 1.352 nmol L ⁻¹ with excellent linearity (R ² = 0.9996) over the range of 5–1000 nmol L ⁻¹ were obtained for Ch and ACh, respectively. The developed analytical methods showed excellent accuracy and precision for the determination of Ch and ACh in human serum samples avoiding their pretreatment or purification. View Show abstract Preparation of highly conductive biochar nanoparticles for rapid and sensitive detection of 17β-estradiol in water Article Sep 2018 ELECTROCHIM ACTA Yang Liu Xinwei Dong Lingzhi He Yunxian Piao We developed a sensitive and cost effective sensor based on highly conductive and adsorptive biochar nanoparticles for electrochemical detection of 17β-estradiol. The biochar is ubiquitous and has the advantages of large specific surface area, cost effectiveness, and good stability, which has the potential to be utilized for electrode construction as long as the conductivities are guaranteed. We obtained nano sized biochar particles (BCNPs) and investigated electrochemical properties of the BCNPs with variation of the pyrolysis conditions. BCNPs with high conductivity (BCNP800) were obtained and utilized for reproducible and stable electrochemical sensor construction. The produced highly conductive BCNP800 sensor had exhibited improved amperometric responses such as decreased impedance, lowered anodic potential and enhanced current signal toward 17β-estradiol compared with the pristine glassy carbon electrode. In addition, the adsorption-accumulation by BCNP800 for 17β-estradiol resulted in great sensing signal amplification. With synergetic effect of high adsorption and conductivity properties of the BCNP800 sensor a detection limit of 11.30 nM was obtained. The accuracy and reliability of the BCNP800 sensor were confirmed to be compatible with the high performance liquid chromatography analysis and it could be successfully applied for real environmental water analysis. View Show abstract Biomass-waste derived graphene quantum dots and their applications Article Aug 2018 CARBON Aumber Abbas Lim Tuti Mariana Anh N. Phan Energy crisis, environmental deterioration and increasing customer needs have compelled scientists to search facile, low cast, and green routes for the production of novel advanced materials from renewable resources. Among various materials explored, carbon based nanomaterials, especially graphene and graphene quantum dots (GQDs), have attracted extensive attention recently owning to their intriguing properties, such as high conductivity, extensive surface area, good biocompatibility, low toxicity, and long life. This review focuses on the conversion of biomass-waste into GQDs through a number of facile, low cost and scalable routes. Factors affecting the physical and chemical properties of GQDs for numerous promising applications have also been discussed. GQDs have shown promising applications in the field of catalysis, carbon fixation, fuel cells, bio-imaging, drug delivery, and gas sensors. Interestingly, the recent and novel applications of GQDs in the conversion and storage of energy has been discussed here. Finally, the remaining challenges, future perspectives and possible research directions in the field are presented. View Show abstract Removal of 17β-estradiol by using highly adsorptive magnetic biochar nanoparticles from aqueous solution Article Jul 2018 CHEM ENG J Xinwei Dong Lingzhi He Hui Hu Yunxian Piao Through biochar nanoparticles (BCNPs) obtained at pyrolysis temperatures of 400 °C, 600 °C and 800 °C, three types of biomass-derived adsorbent, magnetic BCNPs (Mag-BCNP400, Mag-BCNP600 and Mag-BCNP800) were synthesized. The BCNPs possessed large surface area, nearly 14.5 times higher than that of the biochar microparticles, which contributed to enhanced adsorption capacity. Adsorption characteristics of 17β-estradiol (E2) by Mag-BCNPs were investigated and described by a pseudo-second-order model. The thermodynamic study indicated that adsorption was spontaneous and the adsorption isotherms were well described by Langmuir model with the maximum adsorption capacities of 50.24, 41.71 and 34.06 mg/g for Mag-BCNP400, Mag-BCNP600 and Mag-BCNP800, respectively. Fourier transform infrared analysis suggested that hydrophobic interactions and π–π electron donor-acceptor interactions for E2 sorption to Mag-BCNPs may occur simultaneously, whereas the dominating mechanisms may change from hydrophobic interactions to π–π interactions as pyrolysis temperatures increased from 400 °C to 800 °C. Mag-BCNPs can be easily separated using a magnetic force and regenerated by ozonation. Therefore, with the advantageous features of low cost, ease of separation and good reusability, the Mag-BCNPs can be used as potential adsorbents for removing E2 from water. View Show abstract Covalent immobilization of laccase on citric acid functionalized micro-biochars derived from different feedstock and removal of diclofenac Article Jun 2018 CHEM ENG J Linson Lonappan Yuxue Liu Tarek Rouissi Rao Y. Surampalli Immobilization of enzymes on the solid supports can improve the stability as well as catalytic properties of enzymes. In this study, biochar derived from various feedstocks were used as immobilization support considering biochars carbon negative as well as sustainable properties. Partially purified (concentrated) crude laccase was covalently immobilized onto pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) micro-biochars using optimized 5% w/v glutaraldehyde. Moreover, citric acid pretreatment improved the laccase binding capacity of all the micro-biochars and the highest laccase binding of 40.2±1.8 U g-1 was observed with BC-PM in comparison with raw BC-PM (34.1±1.1 U g-1). The enhanced binding of laccase on BC-PM over wood derived biochars was attributed to the higher surface area (46.1 m2 g-1) of BC-PM. Moreover, feedstock selection as well as a method of production influenced the biochar physicochemical properties such as surace area and consequently, different biochars showed various laccase immobilization efficiencies. BC-PW showed better pH, thermal, storage, and operational stability, compared with BC-AS and BC-PM. While applying the laccase bound micro-biochar, complete removal was observed in 2 h under batch mode with 0.5 g of laccase bound BC-PM at an environmentally relevant concentration of 500 µg L-1 in wastewater effluent. About 40% of the laccase activity was retained with all the laccase-bound microbiochars after 5 cycles of diclofenac treatment. View Show abstract Quality of epidemiological studies: Procedural rules for uncertain science for policy, a case study on bisphenol-A Article Jun 2018 ENVIRON SCI POLICY Laura Maxim Jeroen P. van der Sluijs This paper proposes a method for in-depth mapping of heterogeneity in expert judgment, in the evaluation of the quality of epidemiological studies used in regulatory chemical risk assessment. Whereas consensus in scientific advisory groups provides legitimation for subsequent political action, it can also have unintended effects on the quality of regulatory risk assessment. Based on empirical testing of our method, called Qualichem_epi, with ten experts and two epidemiological case studies about bisphenol A (BPA)’s effects on human health, we have shown that expert judgment plays an essential role in managing uncertainty and deciding what “quality” of a study actually means. We found substantial heterogeneity of scientists’ judgments about the quality of epidemiological studies, even if the same criteria were used for the assessment. This heterogeneity is not present anymore in reports produced by expert groups, where results are presented under the collective signature of all the scientists involved. We argue that flattening heterogeneity can be an important problem when it is not the result of true scientific agreement but only a secondary effect of consensus-based working procedures of agencies that experts have to follow. Qualichem_epi provides an easy to understand color-based picture of both majority and minority opinions in a scientific advisory group. We suggest that it could be used on a regular basis for communicating quality assessments of epidemiological studies in regulatory chemical risk assessment. View Show abstract Pinewood nanobiochar: A unique carrier for the immobilization of crude laccase by covalent bonding Article Apr 2018 INT J BIOL MACROMOL Mitra Naghdi Mehrdad Taheran Satinder Kaur Brar Rao Y. Surampalli Nanotechnology-inspired biocatalytic systems attracted attention for many applications since nanosized supports for enzyme immobilization can improve efficiency-determining factors e.g. enhancing the surface area and loading capacity and reducing the mass transfer resistance. Among the nanomaterials, nanobiochar has unique features as a support for enzyme immobilization i.e. high surface to volume ratio, porous structure, and presence of functional groups on its surface. However, the performance of the immobilization is highly dependent on the immobilization conditions and the properties of the enzyme and the support material. In this research, crude laccase was covalently immobilized onto functionalized nanobiochar using a two-step method of diimide-activated amidation. The effect of different parameters was investigated. The optimal conditions were found to be 14 mg/mL of laccase concentration, 5 mg/mL of nanobiochar, 8.2 mM of cross-linker and 3 h of contact time. For investigating the pH, thermal, storage, and operational stability, the sample obtained from the optimized conditions was used. The results showed the higher stability of immobilized laccase against temperature and pH variation compared to free laccase. In addition, immobilized laccase maintained its catalytic performance up to seven cycles of utilization and showed more than 50% of initial activity after two months of room temperature storage. View Show abstract Sorptive removal of phenolic endocrine disruptors by functionalized biochar: Competitive interaction mechanism, removal efficacy and application in wastewater Article Nov 2017 Mohammad Boshir Ahmed John Zhou Huu Hao Ngo Kireesan Sornalingam Sorptive removal of six phenolic endocrine disrupting chemicals (EDCs) estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethynylestradiol (EE2), bisphenol A (BPA) and 4-tert-butylphenol (4tBP) by functionalized biochar (fBC) through competitive interactions was investigated. EDC sorption was pH dependent with the maximum sorption at pH 3.0–3.5 due to hydrogen bonds and π-π interactions as the principal sorptive mechanism. Sorption isotherm of the EDCs was fitted to the Langmuir model. Sorption capacities and distribution coefficient values followed the order E1 > E2 ≥ EE2 > BPA > 4tBP > E3. The findings suggested that EDC sorption occurred mainly through pseudo-second order and external mass transfer diffusion processes, by forming H-bonds along with π-π electron-donor–acceptor (EDA) interactions at different pH. The complete removal of ∼500 μg L−1 of each EDC from different water decreased in the order: deionised water > membrane bioreactor (MBR) sewage effluent > synthetic wastewater. The presence of sodium lauryl sulphonate and acacia gum in synthetic wastewater significantly suppressed sorption affinity of EDCs by 38–50%, hence requiring more fBC to maintain removal efficacy. View Show abstract Combination of Electrochemical Biosensor and Textile Threads: A Microfluidic Device for Phenol Determination in Tap Water Article Jul 2017 BIOSENS BIOELECTRON Emmanuelle A. Carneiro Douglas Agustini e L.H. Marcolino-Junior Fabio Caetano Microfluidic devices constructed using low cost materials presents as alternative for conventional flow analysis systems because they provide advantages as low consumption of reagents and samples, high speed of analysis, possibility of portability and the easiness of construction and maintenance. Herein, is described for the first time the use of an electrochemical biosensor for phenol detection combined with a very simple and efficient microfluidic device based on commercial textile threads. Taking advantages of capillary phenomena and gravity forces, the solution transportation is promoted without any external forces or injection pump. Screen printed electrodes were modified with carbon nanotubes/gold nanoparticles followed by covalent binding of tyrosinase. After the biosensor electrochemical characterization by cyclic voltammetry technique, the optimization of relevant parameters such as pH, potential of detection and linear range for the biosensor performance was carried out; the system was evaluated for analytical phenol detection presenting limit of detection and limit of quantification 2.94 nmol L⁻¹ and 8.92 nmol L⁻¹ respectively. The proposed system was applied on phenol addition and recovery studies in drinking water, obtaining recoveries rates between 90 and 110%. View Show abstract Agricultural biomass-derived magnetic adsorbents: Preparation and application for heavy metals removal Article Jun 2017 J TAIWAN INST CHEM E Noraini M.N Raihan Othman Mubarak N Mujawar Ezzat Chan Abdullah This paper discusses the synthesis of magnetic adsorbents from agricultural waste and their applications in heavy metals removal. The general methods for preparing magnetic adsorbents and the mechanisms of heavy metal sorption are also reviewed in detail. These mechanisms are related to the utilization of magnetic adsorbents, particularly sugarcane bagasse in heavy metals removal, such as nickel, cadmium, lead, and arsenic. Converting sugarcane bagasse into magnetic adsorbents could solve environmental problems, such as agricultural waste and water pollution. A brief summary of the synthesis of magnetic biochar from sugarcane bagasse and its applications in heavy metals removal is also presented. Thus, this study proposes magnetic-based materials as potential candidates for wastewater treatment, and this adds new dimensions to numerous applications of the carbon family. View Show abstract The influence and mechanism of different acid treatment to activated carbon used as air-breathing cathode catalyst of microbial fuel cell Article May 2017 ELECTROCHIM ACTA Zhong Wang Yi Liu Kexun li Jinfeng Lu Commercial activated carbon (AC) was subjected to various kinds of acid treatment, which were employed as air-breathing cathodes for Microbial fuel cells (MFCs). Obviously, both the acid species and the concentration of acidic solution have an effect on the catalytic activity of AC, and the electrochemical performance of AC was increased after acid treatment. Cathodes used 1 M H3PO4 treated AC (AC-H3PO4-1) showed the maximum power density of 1546 ± 43 mW m⁻², which was 115% higher than the pristine AC. The electrochemical studies showed that AC-H3PO4-1 exhibited the least resistance and highest kinetic activity. In addition, Nitrogen adsorption-desorption demonstrated that the acid treatment could increase the total surface area and pore volume, especially the exposure of mesopore to the reactants. Raman, X-ray photoelectron spectroscopy and temperature programmed desorption with ammonia revealed that the degree of graphitization, surface oxygenic functional groups and the acid sites of AC were enhanced after acid treatment. However, too much strong acidic functional groups were detrimental to the oxygen reduction reaction. In general, the treatment of activated carbon with 1 M H3PO4 will be a promising way to improve the performance of microbial fuel cells. View Show abstract Optimized removal of Oxytetracycline and Cadmium from contaminated waters using chemically-activated and pyrolyzed biochars from forest and wood-processing residues Article Sep 2017 Aylin Aghababaei Mohamed Chaker Ncibi Mika Sillanpää In the present investigation, the adsorptive removal of the antibiotic drug oxytetracycline (OTC) and toxic heavy metal cadmium (Cd) from aqueous solution was carried out using forest and wood-processing residues. Numerous biochars were prepared using different chemical agents (H3PO4, H2SO4, NaoH and KOH) and pyrolysis times and temperatures. Several elemental, chemical and structural characterizations were performed. The optimum conditions for pyrolysis to enable the production of biochars with well-developed porosity was 600 °C for 1 h, for both residues. The adsorption process using selected activated biochars was optimized with respect to reaction time, pH, temperature and initial load of pollutants. Under optimized operating conditions, and based on equilibrium modeling data, the biochars which showed the highest removal efficiencies of OTC and cadmium were “5M H3PO4 forest” (263.8 mg/g) and “1M NaoH forest” (79.30 mg/g), respectively. Compared to adsorbents reported in the literature, the efficiencies of those biochars are highly competitive. View Show abstract A promising route of magnetic based materials for removal of Cadmium and Methylene blue from waste water Article Feb 2017 Ruthiraan Manimaran Ezzat Chan Abdullah Mubarak N Mujawar Noraini M.N Adsorption equilibrium is an essential measure to be controlled in attaining the maximum capacity of an adsorbent. Synthesis of magnetic biochar by using various discarded biomass possesses varied adsorbate adsorption capability. Maximising the removal percentage of the pollutant from wastewater by altering operational parameters are the very important skill to be mastered by environmentalist. In this research work, mangosteen peel derived ferric oxide magnetic biochar was pyrolysed at 800 °C for 20 min via modified muffle furnace at zero oxygen environment. The factors affecting adsorption were studied for two different liquid pollutant. A comparative study was carried out for adsorption of methylene blue dye and Cd (II) ions by altering the adsorbate pH, agitation speed, and contact time and particle diffusion mechanism were investigated respectively. The removal of methylene blue dye attained maximum removal at pH of 7.0 and above, 50 min contact time and 150 rpm agitation speed. Moreover, adsorption equilibrium of Cd (II) ions reaches maximum removal percentage at the pH value of 8.0, agitation speed of 150 rpm and 60 min contact time. The thermodynamics study was performed to further understand the effect of operating temperature in determining the adsorption spontaneous of adsorption medium. The experimental analysis reveals that the produced magnetic biochar is viable for adsorption of both heavy metal ions and organic pollutant at optimised condition. View Show abstract Immobilized laccase on oxygen functionalized nanobiochars through mineral acids treatment for removal of carbamazepine Article Jan 2017 SCI TOTAL ENVIRON Mitra Naghdi Mehrdad Taheran Satinder Kaur Brar Rao Y. Surampalli View Gold nanoparticles supported on multi-walled carbon nanotubes produced by biphasic modified method and dopamine sensing application Article Nov 2016 SENSOR ACTUAT B-CHEM Fabio Caetano Leticia B. Felippe Aldo J. G. Zarbin Luiz H. Marcolino-Junior The present work describes the synthesis of a nanocomposite between carbon nanotubes and gold nanoparticles through modified Brust-Schiffin route. Observations by XRD, TEM and TGA techniques showed an uniform, well-distributed and non-agglomerated dispersion of gold nanoparticles (7 ± 4 nm of average size) completely loaded onto MWCNTs surface. After further structural characterizations, the nanocomposite was casted onto a glassy carbon electrode and characterized by cyclic voltammetry. A seven-fold density current enhancement was observed, provided by the high superficial electroactive area of the active material. Sensing properties of so-synthesized nanocomposite were tested for dopamine detection in presence of ascorbic and uric acid by square wave voltammetry. After analytical parameters optimization, the dopamine-sensor presented linear range of 4.8 × 10⁻⁷ to 5.7 × 10⁻⁶ mol L⁻¹ with detection and quantification limits of 7.1 × 10⁻⁸ and 2.4 × 10⁻⁷ mol L⁻¹ respectively and high sensitivity. The proposed method was applied in synthetic cerebrospinal fluids samples and the detection of DA in presence of potentially interferences in real samples was performed with satisfactory results. View Show abstract Protein capped Cu nanoclusters-SWCNT nanocomposite as a novel candidate of high performance platform for organophosphates enzymeless biosensor Article Oct 2016 Hassan Bagheri Abbas Afkhami Hosein Khoshsafar Alireza Shahriyari A biocompatible nanocomposite including bovine serum albumin (BSA) template Cu nanoclusters (CuNCs@BSA) and single-walled carbon nanotubes (SWCNT) was synthesized to fabricate a highly sensitive electrochemical biosensor for paraoxon as a model of organophosphates. The UV-Vis, fluorescence and Fourier transform infrared (FTIR) demonstrated that BSA entrapped in the nanocomposite film have been changed in its secondary structure so that it provided an enzyme like activity attributing to the high electrical conductivity of the entrapped copper nanoclusters. Also, the morphology and structure of prepared nanocomposites were investigated by transmission electronic microscopy (TEM) and scanning electron microscopy (SEM). In the prepared nanocomposite, the CuNCs@BSA found to play as a conductive holder as well as an accumulator of redox active centers on the surface of the electrode, and SWCNT improves the electrocatalytic activity along with conductivity of glassy carbon electrode (GCE) surface. The fabricated biosensor exhibited excellent sensitivity, acceptable stability, fast response, and high electrocatalytic activity toward the reduction of paraoxon. The reduction peak current vs paraoxon concentration was linear over the range 50 nM to 0.5 μM and 0.5 to 35 μM, with a limit of detection of 12.8 nM. Notable electrocatalytic properties of the developed electrode toward paraoxon indicated that the nanocomposite possesses a promising potential to fabricate the third generation enzyme-free electrochemical biosensors, bioelectronics and state-of-the-art biomedical devices in the future. View Show abstract Electrochemically reduced graphene and iridium oxide nanoparticles for inhibition-based angiotensin-converting enzyme inhibitor detection Article Jul 2016 Sevinc Kurbanoglu Lourdes Rivas Sibel A. Ozkan Arben Merkoçi View Precipitated and chemically-crosslinked laccase over polyaniline nanofiber for high performance phenol sensing Article Aug 2015 Jae Hyun Kim Sung-gil Hong Ho Jin Sun Jungbae Kim The present study aims at fabricating a laccase (LAC) based amperometric biosensor for detection of phenolic compounds. LAC was immobilized into the porous matrix of polyaniline nanofibers (PANFs) in a three-step process, consisting of enzyme adsorption, precipitation, and crosslinking (EAPC). Immobilized LAC on PANF in the form of EAPC was highly active and stable when compared to control samples of 'enzyme adsorption (EA)' and 'enzyme adsorption and crosslinking (EAC)' samples. For example, the activity of EAPC was 19.7 and 15.1 times higher than those of EA and EAC per unit weight of PANF, respectively. After 6days at room temperature, EAPC maintained 100% of its initial activity, while EA and EAC retained only 7.7% and 11% of their initial activities, respectively. When the samples were subjected to the heat treatment at 60°C over 3h, EAPC maintained 74% of its initial activity, while EA and EAC retained around 1% of their initial activities, respectively. To demonstrate the feasible application of EAPC in biosensors, the enzyme electrodes were prepared and used for detection of phenolic compounds, which are environmentally hazardous chemicals. The sensitivities of biosensors with EA, EAC, and EAPC were 20.3±5.9, 26.6±5.4 and 518±11μAmM(-1)cm(-2), respectively. At 50°C for 5h, EAPC electrode maintained 80% of its initial sensitivity, while EA and EAC electrode showed 0% and 19% of their initial sensitivities, respectively. Thus, LAC-based biosensor using EAPC protocol with PANFs showed a great promise for developing a highly sensitive and stable biosensor for detection of phenolic compounds. Copyright © 2015 Elsevier Ltd. All rights reserved. View Show abstract Tyrosinase conjugated reduced graphene oxide based biointerface for bisphenol A sensor Article Jul 2015 K.Kamil Reza Md Azahar Ali SaurabhSrivastava Ashok Manikrao Biradar View Fluorescence polarization immunoassay method for bisphenol A residue in environmental water samples based on monoclonal antibody and 4’-(Aminomethyl) fluorescein Article Apr 2015 Anal Methods Pinong Huang Suqing Zhao Sergei A. Eremin Bin Guo Based on a sensitive monoclonal antibody against bisphenol A (BPA) and a new tracer named BVA-AMF, a homogeneous fluorescence polarization immunoassay (FPIA) was developed and applied in the determination of bisphenol A in environmental water samples. BVA was selected as the hapten to couple with bovine serum albumin and the conjugate was used as the immunogen for the monoclonal antibody production. Three fluorescein-labeled BVA tracers with different structures (BVA-AMF, BVA-EDF, BVA-lysFITC) were synthesized. Under the same optimal conditions, BVA-AMF showed most sensitivity for FPIA and the detection of BPA was with a limited detection of 5.60 ng/mL, IC50 of 140 ng/mL and a dynamic range of 11.32-904.21 ng/mL approximately. In this assay, several similar compounds were shown of little significantly with the cross reactivity being less than 0.15%. Four different kinds of water samples were analyzed, with recoveries being 87.91%-114.28%. The detection standard curve for BPA exhibited good linearity (R2=0.9913, n=3). Compared with ELISA and HPLC methods, FPIA showed reliability and high correlation with ELISA of 0.9964 and HPLC of 0.9971. The immunoassay technique demonstrated that was responsible for detection of BPA in authentic environmental water samples. View Show abstract Recent advances in utilization of biochar Article Feb 2015 RENEW SUST ENERG REV Kezhen Qian Kumar Ajay Hailin Zhang R. Huhnke View Laccase biosensor based on screen-printed electrode modified with thionine–carbon black nanocomposite, for Bisphenol A detection Article Oct 2013 ELECTROCHIM ACTA Marianna Portaccio Daniela Di Tuoro Fabiana Arduini Maria Lepore The relevance of Bisphenol A (BPA) in human health is well-known. For this reason we designed and developed a biosensor based on a bionanocomposite (laccase-thionine-carbon black)-modified screen-printed electrode. Thionine, a commercially available dye, was used as electrochemical mediator coupled with a nanostructured carbon black. By means of cyclic voltammetry, the interaction of thionine adsorbed on modified screen printed electrode with laccase/BPA reaction products has been studied. In addition, the immobilization of laccase by physical adsorption on the surface of thionine-carbon black modified screen printed electrodes was investigated. The response of the biosensor has been optimized in terms of enzyme loading, pH and applied potential reaching a linear concentration range of 0.5-50 mu M, a sensitivity of 5.0 +/- 0.1 nA/mu M and a limit-of-detection (LOD) of 0.2 mu M. The developed biosensor has been also challenged in tomato juice samples contained in metallic cans where release of BPA due to the epoxy resin coating can be assumed. A satisfactory recovery value comprised between 92% and 120% was obtained. View Show abstract 3D metal-organic framework as highly efficient biosensing platform for ultrasensitive and rapid detection of bisphenol A Article Oct 2014 Xue Wang XB Lu Lidong Wu Jiping Chen View Functional graphene-gold nano-composite fabricated electrochemical biosensor for direct and rapid detection of bisphenol A Article Jan 2015 ANAL CHIM ACTA Daodong Pan Yuanyuan Gu Hangzhen Lan Huiju Gao In this research, the graphene with excellent dispersity is prepared successfully by introducing gold nanoparticle to separate the individual sheets. Various techniques are adopted to characterize the prepared graphene and graphene-gold nanoparticle composite materials. This fabricated new composite material is used as the support material to construct a novel tyrosinase based biosensor for detection of bisphenol A (BPA). The electrochemical performances of the proposed new enzyme biosensor were investigated by differential pulse voltammetry (DPV) method. The proposed biosensor exhibited excellent performance for BPA determination with a wide linear range (2.5×10(-3)-3.0μM), a highly reproducible response (RSD of 2.7%), low interferences and long-term stability. And more importantly, the calculated detection limit of the proposed biosensor was as low as 1nM. Compared with other detection methods, this graphene-gold nanoparticle composite based tyrosinase biosensor is proved to be a promising and reliable tool for rapid detection of BPA for on-site analysis of emergency BPA related pollution affairs. Copyright © 2014 Elsevier B.V. All rights reserved. View Show abstract Effect of pyrolysis temperature on carbon obtained from green tea biomass for superior lithium ion battery anodes Article Oct 2014 CHEM ENG J Sang-Wook Han Dong-Won Jung Jae-Hun Jeong Eun-Suok Oh Carbonaceous materials pyrolyzed from green tea leaves are fabricated and characterized for their potential application as high-performance anodes in lithium ion batteries (LIBs). Three different pyrolysis temperatures (700, 800, and 900 °C) are employed, and the most efficient pyrolysis temperature is determined through a variety of physical and electrochemical measurements. The carbon pyrolyzed at a relatively low temperature of 700 °C contains numerous functional groups, defects that are different from those in graphitic carbon, and large pores. Consequently, the sample exhibited a relatively large capacity of 471 mAh g-1 at the 50th cycle, even though high initial irreversibility was observed. Furthermore, when compared to the extremely low capacity of graphite (12.7 mAh g-1), the carbon specimen pyrolyzed at 700 °C displays an excellent high-rate capability of 131 mAh g-1 at 10 C. Such a result is attributed to the relatively isotropic structures and large-size pores in the sample, which facilitates the rapid diffusion of lithium ions. View Show abstract Highly Conductive Graphite Nanoparticle Based Enzyme Biosensor for Electrochemical Glucose Detection Article Apr 2014 SENSOR ACTUAT B-CHEM Dong Ju Han Tae Seok Seo Yunxian Piao View Analytical Methods for the Quantification of Bisphenol A, Alkylphenols, Phthalate Esters, and Perfluoronated Chemicals in Biological Samples Article Jan 2014 ANAL SCI Hiroyuki Nakazawa Yusuke Iwasaki Rie Ito Our modern society has created a large number of chemicals that are used for the production of everyday commodities including toys, food packaging, cosmetic products, and building materials. We enjoy a comfortable and convenient lifestyle with access to these items. In addition, in specialized areas, such as experimental science and various medical fields, laboratory equipment and devices that are manufactured using a wide range of chemical substances are also extensively employed. The association between human exposure to trace hazardous chemicals and an increased incidence of endocrine disease has been recognized. However, the evaluation of human exposure to such endocrine disrupting chemicals is therefore imperative, and the determination of exposure levels requires the analysis of human biological materials, such as blood and urine. To obtain as much information as possible from limited sample sizes, highly sensitive and reliable analytical methods are also required for exposure assessments. The present review focuses on effective analytical methods for the quantification of bisphenol A (BPA), alkylphenols (APs), phthalate esters (PEs), and perfluoronated chemicals (PFCs), which are chemicals used in the production of everyday commodities. Using data obtained from liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analyses, assessments of the risks to humans were also presented based on the estimated levels of exposure to PFCs. View Show abstract Amperometric biosensor for bisphenol A based on a glassy carbon electrode modified with a nanocomposite made from polylysine, single walled carbon nanotubes and tyrosinase Article Aug 2013 Matthew Han Shiqin Chen Ying Qu Chunya Li We have prepared a nanocomposite consisting of single-walled carbon nanotubes and polylysine. It was characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, and by UV/vis and FTIR spectroscopy. Tyrosinase was covalently immobilized on the nanocomposite, and the resulting bioconjugate deposited on a glassy carbon electrode to form a biosensor for bisphenol A. The biosensor was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. Under optimized experimental conditions, the biosensor gives a linear response to bisphenol A in the 4.00 nM to 11.5 μM concentration range. Its sensitivity is 788 mA M−1 cm−2, and the lower detection limit is 0.97 nM (at an S/N of 3). The biosensor shows good repeatability, reproducibility and long-term stability. In a preliminary practical application, it was successfully applied to the determination of bisphenol A in leachates of plastic spoons. Figure Single-walled carbon nanotubes-polylysine (SWCNT-PLL) nanocomposite was prepared and thoroughly characterized. The obtained nanocomposite was used as a platform to immobilize tyrosinase (Tyr) onto a glassy carbon electrode (GCE) to fabricate a biosensor for bisphenol A (BPA) View Show abstract Sensitive amperometric biosensor for phenolic compounds based on graphene-silk peptide/tyrosinase composite nanointerface Article Jan 2013 Ying Qu Ming Ma Zhengguo Wang Chunya Li New graphene-silk peptide (Gr-SP) nanosheets were prepared and successfully fabricated with tyrosinase (Tyr) as a novel biosensor for the determination of phenolic compounds. The Gr-SP nanosheets were fully characterized with transmission electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy, UV/Vis and FTIR spectra. The developed biosensors were also characterized with scanning electronic microscopy and electrochemical impedance spectroscopy. Using bisphenol A (BPA) as a model substrate in the sensing system, a number of key factors including the volume of Gr-SP-Tyr solution, the applied potential, pH values, temperature, and the Tyr/Gr-SP ratio that influence the analytical performance of the biosensor were investigated. The biosensor gave a linear response on the concentration ranges of 0.001-16.91μM for catechol with the sensitivity of 7634mAM(-1)cm(-2), 0.0015-21.12μM for phenol with the sensitivity of 4082mAM(-1)cm(-2), and 0.002-5.48μM for BPA with the sensitivity of 2511mAM(-1)cm(-2). The low detection limits were estimated to be 0.23, 0.35 and 0.72nM (S/N=3) for catechol, phenol and BPA, respectively. The biosensors also exhibit good repeatability and long-term stability. The practical application of the biosensor was also demonstrated by the determination of BPA leaching from commercial plastic drinking bottles. View Show abstract Fullerene-C 60 sensor for ultra-high sensitive detection of bisphenol-A and its treatment by green technology Article Jan 2013 SENSOR ACTUAT B-CHEM Jahangir Ahmad Rather Karolien De Wael Available online xxx Keywords: Bisphenol-A (BPA) Fullerene sensor (C60/GCE) Endocrine disruptor Green technology a b s t r a c t Endocrine disruptors (EDCs) are environmental pollutants that, once incorporated into an organism, affect the hormonal balance of humans and various species. Its presence in environment is of great importance in water quality related questions. The proposed method describes the development of an accurate, sen-sitive and selective sensor for the detection of bisphenol-A (BPA) and its treatment by green technology. A fullerene (C 60) fabricated electrochemical sensor was developed for the ultrasensitive detection of BPA. The homemade sensor was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and chronocoulometry. The influence of measuring parameters such as pH and C 60 loading on the analytical performance of the sensor was evaluated. Various kinetic parameters such as electron transfer number (n); charge transfer coefficient (˛); electrode surface area (A) and diffusion coefficient (D) were also calculated. Under the optimal conditions, the oxidation peak current was linear over the concentration range of 74 nM to 0.23 M with the detection limit (LOD) of 3.7 nM. The fabricated sen-sor was successfully applied to the determination of BPA in wastewater samples and it has promising analytical applications for the direct determination of BPA at trace level. View Show abstract Effects of carbon nanotube functionalization on the mechanical and thermal properties of epoxy composites Article Jun 2009 CARBON Kai Yang Mingyuan Gu Yiping Guo Guohong Mu Chemically functionalized multi-walled carbon nanotube (MWCNT)/bisphenol-A glycidol ether epoxy resin/2-ethyl-4-methylimidazole composites were prepared. MWCNTs were first treated by a 3:1 (v/v) mixture of concentrated H2SO4/HNO3, and then triethylenetetramine (TETA) grafting was carried out. X-ray photoelectron spectroscopy analysis proved the effectiveness of H2SO4/HNO3 treatment and confirmed the TETA functionalization mechanism. Chemical functionalization decreases the crystalline content of MWCNTs, however, it did not greatly disrupt their structure. Transmission electron microscopy showed that there was a TETA thin layer on the MWCNT surface, which contributes to the homogenous dispersion of MWCNTs in epoxy matrix and the improvement of the MWCNT–epoxy interfacial interaction. Thus the impact strength, bending strength and thermal conductivity of the composites are enhanced. View Show abstract Nano-graphene-based tyrosinase biosensor for rapid detection of bisphenol A Article Mar 2012 Lidong Wu Dehui Deng Jing Jin Jiping Chen Hydrophilic nanographene (NGP) prepared by ball milling of graphite was used as the support to construct a novel tyrosinase biosensor for determination of bisphenol A (BPA). The performances of the nanographene-based tyrosinase biosensor were systematically compared with those of multiwall carbon nanotubes (MWNTs) modified tyrosinase biosensors. The results indicated that the nanographene-based tyrosinase biosensor provided significant advantages over MWNTs-based tyrosinase biosensor in term of response, repeatability, background current and limit of detection (LOD), which could be attributed to its larger specific surface area and unique hierarchical tyrosinase-NGP nanostructures. The nanographene-based tyrosinase biosensor displayed superior analytical performance over a linear range from 100 nmol L(-1) to 2000 nmol L(-1), with LOD of 33 nmol L(-1) and sensitivity of 3108.4 mA cm(-2)M(-1). The biosensor was further used for detecting BPA (leaching from different vessels) in tap water, and the accuracy of the results was validated by high performance liquid chromatography (HPLC). The nanographene-based tyrosinase biosensor proved to be a promising and reliable tool for rapid detection of BPA leached from polycarbonate plastic products and for on-site rapid analysis of emergency pollution affairs of BPA. View Show abstract Bisphenol A: An endocrine disruptor with widespread exposure and multiple effects Article May 2011 Beverly S Rubin Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. This compound is a building block of polycarbonate plastics often used for food and beverage storage, and BPA is also a component of epoxy resins that are used to line food and beverage containers. Studies have shown that BPA can leach from these and other products in contact with food and drink, and as a result, routine ingestion of BPA is presumed. This compound is also found in an enormous number of other products that we come into contact with daily, and therefore it is not surprising that it has been detected in the majority of individuals examined. BPA is a known endocrine disruptor. Although initially considered to be a weak environmental estrogen, more recent studies have demonstrated that BPA may be similar in potency to estradiol in stimulating some cellular responses. Moreover, emerging evidence suggests that BPA may influence multiple endocrine-related pathways. Studies in rodents have identified adverse effects of BPA at levels at or below the current acceptable daily intake level for this compound. The various reported adverse effects of BPA are reviewed, and potential mechanisms of BPA action are discussed. Much more investigation is needed to understand the potential adverse health effects of BPA exposure in humans and to understand the multiple pathways through which it may act. Although many questions remain to be answered, it is becoming increasingly apparent that exposure to BPA is ubiquitous and that the effects of this endocrine disruptor are complex and wide-ranging. View Show abstract GC-MS analysis of bisphenol A in human placental and fetal liver samples Article Jan 2011 Jie Zhang Gerard M Cooke Ivan Curran Xu-Liang Cao A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography-mass spectrometry (GC-MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings. View Show abstract Amperometric catechol biosensor based on polyaniline-polyphenol oxidase Article Mar 2010 Yongyan Tan Xiaoxia Guo Jinghui Zhang Jinqing Kan A novel catechol biosensor was described based on the immobilization of polyphenol oxidase (PPO) into polyaniline (PANI), which was easily constructed by direct electropolymerization of aniline in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate (EMIES). The developed biosensor for the detection of catechol has a linear range of 1.25-150 micromol dm(-3). The maximum response current (I(max)) and the Michaelis-Menten constant (k'(m)) are 0.62 microA and 146 micromol dm(-3), respectively. The activation energy (E(a)) of the PPO catalytic reaction is 31.1 kJ mol(-1) in the B-R buffer. The biosensor shows good reproducibility (a relative standard deviation of 3.1% was obtained) and remarkable long-term stability (it retains 75% of the original activity after four months). The effects of potential and pH on the response current of the biosensor are also described. View Show abstract Determination of bisphenol A (BPA) in plasma of hemodialysis patients using three methods: LC/ECD, LC/MS, and ELISA Article Nov 2008 TOXICOL MECH METHOD Junko Sajiki Yasuyuki Hasegawa Hiroyuki Hashimoto Tatsuya Morigami ABSTRACT Bisphenol A (BPA) values were compared in plasma of hemodialysis patients and in recycling solvents using LC/MS, LC/electrochemical detector (ECD), and enzyme linked immunosorbent assay (ELISA). BPA values in the plasma and the solvent were 0-8.4 ng/ml and 0-0.8 ng/ml for LC/ECD, 0-4.9 ng/ml and 0-0.8 ng/ml for LC/MS, and 0-15.5 ng/ml and 0-3.1 ng/ml for ELISA, respectively. There was no significant difference among BPA values both in the plasma and the solvents using three methods. Single correlation coefficients between LC/ECD and LC/MS, LC/ECD and ELISA, and LC/MS and ELISA were, respectively, 0.373 (p < 0.002), 0.347 (p < 0.002), and 0.945 (p < 0.001) in the plasma (n = 68-109) and 0.916 (p < 0.001), 0.431 (p > 0.05), and 0.332 (p > 0.05) in the solvents (n = 19). An unknown substance present in the plasma of patients but not healthy volunteers influenced the LC/ECD values of plasma repeated freezing and thawing. The results indicate that LC/MS and ELISA are appropriate for BPA analysis in plasma and both LC/MS and LC/ECD in the recycling solvents and handling with plasma before analysis is important to the analysis of BPA in patients' plasma using LC/ECD. View Show abstract Multiplexed immunoassay using the stabilized enzymes in mesoporous silica Article Sep 2009 Yunxian Piao Dohoon Lee J. Lee Hun-Sung Kim Multiplexed immunoassay system was developed using the enzyme-immobilized mesoporous silica in a form of nanoscale enzyme reactors (NERs), which improve the enzyme loading, activity, and stability. Glucose oxidase (GO) and trypsin (TR) were adsorbed into mesoporous silica and further crosslinked for the construction of NERs, and antibody-conjugated NERs were employed for the analysis of target antigens in a sandwich-type magnetic bead-based immunoassay. This approach, called as NER-LISA (NER-linked immunosorbent assay), generated signals out of enzyme reactions that correlated well with the concentration of target antigens. The detection limit of NER-LISA using NER-GO and anti-human IgG was 67pM human IgG, and the sensitivity was 20 times higher than that of the conventional ELISA using anti-human IgG conjugated GO. Antibody-conjugated NER-GO and NER-TR were successfully employed for the simultaneous detection of two target antigens (human IgG and chicken IgG) in a solution by taking advantage of signals at different wavelengths (absorbances at 570nm and 410nm, respectively) from the assays of GO and TR activities, demonstrating the potential of NER-LISA in multiplexed immunoassay. The NER-LISA approach also enabled the successful use of a protease (trypsin), because the NER approach can effectively retain the protease molecules within the mesoporous silica and prevent the digestion of antibodies and enzymes during the whole process of NER-LISA. View Show abstract pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase Article Apr 2007 Frida Jacobson Arthur Pistorius Daniel Farkas Richard Neutze Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity. View Show abstract Humic acid coated Fe3O4 magnetic nanoparticles as highly efficient Fenton-like catalyst for complete mineralization of sulfathiazole Jan 2011 559 Zhang Recommendations Discover more about: Bisphenol A Article Nanographene-tyrosinase-chitosan-based Electrochemical Enzyme Biosensor for Rapid Detection of Bisph... December 2014 · Modern Food Science and Technology Yangying Sun Y.-Y. Gu D.-D. Pan [...] Xiaoqun Zeng In this study, an electrochemical, graphene-based biosensor was developed for rapid detection of trace amounts of bisphenol A (BPA). Graphene was prepared by chemical reduction, and its structure and morphology were characterized by using transmission electron microscopy and selected area electron diffraction. Subsequently, graphene, tyrosinase, and chitosan were mixed in an optimized ratio to ... [Show full abstract] construct the graphene-tyrosinase-chitosan-modified electrode. Cyclic voltammetry showed that the modified electrode had good electrochemical characteristics. When tested with 0.1 mol·L-1 phosphate buffered saline (pH 7.0), a wide response range for the detection of BPA was obtained with this biosensor, with a low detection limit. The linear range was from 7.5×10-8 to 2.5×10-6 mol·L-1, the correlation coefficient R2 was 0.996, and the detection limit was 2.18×10-8 mol·L-1 (S/N=3). The results of the stability, reproducibility, and interference tests indicated that the enzyme biosensor prepared in this study exhibited good stability, reproducibility, and high specificity for the detection of BPA. This study provides a sensitive, rapid, and simple method to detect BPA residues. ©, 2014, South China University of Technology. All right reserved. Read more Looking for the full-text? You can request the full-text of this article directly from the authors on ResearchGate. Request full-text Already a member? Log in ResearchGate iOS App Get it from the App Store now. Install Keep up with your stats and more Access scientific knowledge from anywhere or Discover by subject area Recruit researchers Join for free Login Email Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google Welcome back! Please log in. 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